Gene-editing systems for modifying a scn9a or scn10a gene and methods of use thereof

ABSTRACT

Disclosed herein are highly efficient gene-editing systems for editing a voltage-gated sodium channel gene, such as sodium voltage-gated channel alpha subunit 9 (SCN9A) or sodium voltage-gated channel alpha subunit 10 (SCN10A), either in vitro or in vivo. The gene-editing systems disclosed herein comprise RNA-guided DNA endonuclease and specific guide RNAs. Also provided herein are uses of the gene-editing systems to modify the target gene, thereby alleviating pain.

RELATED APPLICATION

This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application No. 62/833,523, filed Apr. 12, 2019, the entire contents of which are incorporated herein by reference.

BACKGROUND

Gene editing (including genomic editing) is a type of genetic engineering in which nucleotide(s)/nucleic acid(s) is/are inserted, deleted, and/or substituted in a DNA sequence, such as in the genome of a targeted cell. Recent gene editing strategies which utilize RNA-guided endonucleases, such as Cas9, enable site-specific DNA modification; however, it has been found that not all RNA-guided endonuclease, guide RNA pairs edit with high efficiency. Therefore, there still remains a critical need for identifying effective RNA-guided endonuclease, guide RNA pairs that effectively modify a gene of interest.

SUMMARY

The present disclosure is based, at least in part, on the development of efficient gene editing systems for modifying a voltage-gated sodium channel gene, such as sodium voltage-gated channel alpha subunit 9 (SCN9A) or sodium voltage-gated channel alpha subunit 10 (SCN10A). In some embodiments, the gene editing system relies on the identification of pairs of effective RNA-guided endonuclease and guide RNAs (e.g., those disclosed herein) for effective modification of a voltage-gated sodium channel gene with low off target occurrence.

As such, in some aspects, the disclosure relates to gene-editing systems for modifying a voltage-gated sodium channel gene, such as SCN9A or SCN10A. Such a gene-editing system may comprise: (a) a first polynucleotide moiety, which comprises a first nucleotide sequence encoding a RNA-guided DNA endonuclease, or the RNA-guided DNA endonuclease; and (b) a second polynucleotide moiety, which comprises a second nucleotide sequence encoding a guide RNA (gRNA).

In some embodiments, the gene-editing system may modify a SCN9A gene and comprise: (a) a first polynucleotide moiety, which comprises a first nucleotide sequence encoding a RNA-guided DNA endonuclease, or the RNA-guided DNA endonuclease; and (b) a second polynucleotide moiety, which comprises a second nucleotide sequence encoding a guide RNA (gRNA), wherein the gRNA comprises the nucleotide sequence of any one of SEQ ID NOs: 1-20. A polynucleotide moiety as used herein can be an independent nucleic acid molecule. Alternatively, a polynucleotide moiety can be a portion of a nucleic acid molecule, which may contain one or more additional polynucleotide moieties.

A RNA-guided endonuclease of such a gene-editing system may be Staphylococcus pyogenes (SpCas9), which may be paired with a gRNA comprising the nucleotide sequence of any one of SEQ ID NOs: 1-10. Alternatively or in addition, a RNA-guided endonuclease of such a gene-editing system may be Staphylococcus aureus Cas9 (SaCas9), which may be paired with a gRNA comprising the nucleotide sequence of any one of SEQ ID NOs: 11-20.

In some embodiments, the gene-editing system may modify a SCN10A gene and comprise: (a) a first polynucleotide moiety, which comprises a first nucleotide sequence encoding a RNA-guided DNA endonuclease, or the RNA-guided DNA endonuclease; and (b) a second polynucleotide moiety, which comprises a second nucleotide sequence encoding a guide RNA (gRNA), wherein the gRNA comprises the nucleotide sequence of any one of SEQ ID NOs: 21-40. A RNA-guided endonuclease of such a gene-editing system may be SpCas9, which may be paired with a gRNA comprising the nucleotide sequence of any one of SEQ ID NOs: 21-30. Alternatively or in addition, a RNA-guided endonuclease of such a gene-editing system may be SaCas9, which may be paired with a gRNA comprising the nucleotide sequence of any one of SEQ ID NOs: 31-40.

In some embodiments, the first nucleotide sequence encoding the RNA-guided DNA endonuclease in (a) may further comprise a nucleotide sequence encoding a nuclear localization signal (NLS), which is fused in-frame with the RNA-guided DNA endonuclease. In some embodiments, the NLS is a SV40 NLS.

In some embodiments, the second nucleotide sequence in (b) may further comprise a scaffold sequence. In some examples, the scaffold sequence may be recognizable by SaCas9. Such a scaffold sequence may comprise the nucleotide sequence of GUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUC UCGUCAACUUGUUGGCGAGAUUUU (SEQ ID NO: 41). In other examples, the scaffold sequence may be recognizable by SpCas9. It should be understood that because the second nucleotide sequence encoding the gRNA can be either a DNA sequence or a RNA sequence, any of the uracils (U) in this sequence may be replaced with a thymine (T).

In some embodiments, the first polynucleotide moiety of (a) and the second polynucleotide moiety of (b) are different polynucleotides, at least one of which may be a vector. A vector may be a viral vector, for example an adeno-associated viral (AAV) vector. In some embodiments, the first polynucleotide moiety of (a) and the second polynucleotide moiety of (b) are different AAV vectors.

In some embodiments, a single polynucleotide comprises the first polynucleotide moiety of (a) and the second polynucleotide moiety of (b). The single polynucleotide may be a vector, which may be a viral vector such as an AAV vector. In some embodiments, the AAV is AAV1.

Also within the scope of the present disclosure are nucleic acids and viral particles or sets of viral particles, which collectively comprise any of the gene-editing systems disclosed herein. In some embodiments, the viral particle is, or set of viral particles are, AAV particle(s).

In yet other aspects, the disclosure relates to methods of editing a voltage-gated sodium channel gene, such as SCN9A or SCN10A, the method comprises contacting a cell with: (i) any of the gene-editing systems disclosed herein; (ii) a nucleic acid comprising the gene-editing system; or (iii) a viral particle or a set of viral particles, which collectively comprise the gene-editing system.

In some embodiments, the contacting step is performed by administering the gene-editing system of (a), the nucleic acid of (b), or the viral particle(s) of (c) to a subject in need thereof. In some embodiments, the subject is a human patient having pain.

In some embodiments, the cell is an autologous cell. Alternatively a cell may be a heterologous cell. In some embodiments, the cell is a stem cell, for example an iPSC cell or mesenchymal stem cell. In some examples, the method may further comprise administering the cell with the edited gene to a subject in need thereof (e.g., a human patient having pain).

Also within the scope of the present disclosure are uses of any of the gene-editing systems described herein or components thereof for treating pain, as well as uses thereof for manufacturing a medicament for the intended medical treatment.

The details of one or more embodiments of the disclosure are set forth in the description below. Other features or advantages of the present disclosure will be apparent from the detailed description of several embodiments and also from the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. It is to be understood that the data illustrated in the drawings in no way limit the scope of the disclosure.

FIGS. 1A-1D depict on target editing efficiency of 40 prioritized gRNAs in different cell models. Prioritized gRNAs included (FIG. 1A) ten gRNAs for SpCas9 targeting SCN9A, (FIG. 1B) ten gRNAs for SpCas9 targeting SCN10A, (FIG. 1C) ten gRNAs for SaCas9 targeting SCN9A, and (FIG. 1D) ten gRNAs for SaCas9 targeting SCN10A. These gRNAs were screened in iPSCs, iPSCs stably expressing Cas9, and iPSC-derived sensory neurons. Values represent mean±standard deviation.

DETAILED DESCRIPTION

Gene editing (including genomic editing) is a type of genetic engineering in which nucleotide(s)/nucleic acid(s) is/are inserted, deleted, and/or substituted in a DNA sequence, such as in the genome of a targeted cell. Targeted gene editing enables insertion, deletion, and/or substitution at pre-selected sites in the genome of a targeted cell (e.g., in a targeted gene or targeted DNA sequence). When a sequence of an endogenous gene is edited, for example by deletion, insertion or substitution of nucleotide(s)/nucleic acid(s), the endogenous gene comprising the affected sequence may be knocked-out or knocked-down due to the sequence alteration. Therefore, targeted editing may be used to disrupt endogenous gene expression. Alternatively or in addition, a desired nucleic acid may be inserted into a target site in a DNA sequence (e.g., in an endogenous gene), which is known as targeted integration. “Targeted integration” refers to a process involving insertion of one or more exogenous sequences, with or without deletion of an endogenous sequence at the insertion site. Targeted integration can result from targeted gene editing when a donor template containing an exogenous sequence is present.

The present disclosure is based, at least in part, on the development of efficient gene editing systems for modifying a voltage-gated sodium channel gene, such as sodium voltage-gated channel alpha subunit 9 (SCN9A) or sodium voltage-gated channel alpha subunit 10 (SCN10A). Sodium channels are integral membrane proteins that form ion channels through a cell's membrane. Voltage-gated sodium channels are sodium channels that are “opened” (i.e., allow the flow of sodium ions through the channel) in response to a voltage change. An alpha subunit of a sodium channel forms the core of the channel and is functional on its own (i.e., in the absence of any corresponding beta subunits or other accessory proteins). The family of sodium voltage-gated channels has nine members. The alpha subunits of these channels are Na_(v)1.1, Na_(v)1.2, Na_(v)1.3, Na_(v)1.4, Na_(v)1.5, Na_(v)1.6, Na_(v)1.7, Na_(v)1.8, and Na_(v)1.9, encoded by SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN8A, SCN9A, SCN10A, and SCN11A, respectively.

Na_(v)1.7 (encoded by SCN9A) is expressed, for example, in the dorsal root ganglion, the trigeminal ganglion, and the sympathetic ganglion neurons. Na_(v)1.8 (encoded by SCN10A) is expressed, for example, in the dorsal root ganglion, in unmyelinated small-diameter sensory neurons called C-fibres. Both Na_(v)1.7 and Na_(v)1.8 are involved in nociception (i.e., a sensory mechanism that provides signals that lead to the sensation of pain).

Editing the SCN9A and/or SCN10A gene using any of the methods described herein may be used to treat, prevent and/or mitigate the symptoms of diseases and disorders such as, but not limited to, Congenital Pain Insensitivity, Anosmia, As If Personality, Borderline Personality Disorder, Malignant neoplasm of breast, Non-Small Cell Lung Carcinoma, Cold intolerance, Febrile Convulsions, Diabetes, Diabetes Mellitus, Dissociative disorder, Epilepsy, Erythromelalgia, Primary Erythermalgia, Facial Pain, Herpesviridae Infections, Hereditary Sensory Autonomic Neuropathy Type 5, Hyperplasia, Neuralgia, Hereditary Sensory and Autonomic Neuropathies, Degenerative polyarthritis, Pain, Pain in limb, Postoperative Pain, Parkinson Disease, Postherpetic neuralgia, Prostatic Neoplasms, Pruritus, Seizures, Somatoform Disorder, Tobacco Use Disorder, Trigeminal Neuralgia, Synovial Cyst, Chronic pain, Acute onset pain, Paramyotonia Congenita (disorder), Malaise, Sensory Discomfort, Burning Pain, Indifference to pain, Inflammatory pain, Mechanical pain, Scalp pain, Hereditary Motor and Sensory Neuropathy Type II, Common Migraine, Absence of pain sensation, Malignant neoplasm of prostate, Pain Disorder, Knee Osteoarthritis, Neuropathy, Complex Regional Pain Syndromes, Tonic-clonic seizures, Inherited neuropathies, Prostate carcinoma, Breast Carcinoma, Infantile Severe Myoclonic Epilepsy, Myxoid cyst, Channelopathies, Paroxysmal Extreme Pain Disorder, Painful Neuropathy, Compressive Neuropathies, Congenital Indifference to Pain Autosomal Recessive, Generalized Epilepsy With Febrile Seizures Plus Type 2, Generalized Epilepsy With Febrile Seizures Plus 7, Febrile Seizures Familial 3B, and Small Fiber Neuropathy (Adult-onset is referred to as small fiber neuropathy).

Mutations in the SCN9A gene are known to cause pain perception disorders, including Primary Erythermyalgia, Paroxysmal Extreme Pain Disorder, Congenital Insensitivity to Pain, and Small Fiber Neuropathy. Gain-of-function mutations in the SCN9A gene result in spontaneous pain as observed in Primary Erythermyalgia and Paroxysmal Extreme Pain Disorder. Thus, knock-out or knock-down of the SCN9A gene in patients having Primary Erythermyalgia or Paroxysmal Pain Disorder can be used to treat, prevent and/or mitigate the associated symptoms.

Primary Erythromelalgia is a rare autosomal dominant disorder characterized by episodes of burning pain in the feet and hands in response to heat and movement. Affected individuals typically develop signs and symptoms in early childhood, although in milder cases symptoms can appear later in life. Management of this condition is mainly symptomatic. Besides avoidance of pain triggers (such as heat, exercise, and alcohol), treatment options include cooling and elevating the extremity, use of anesthetics such as lidocaine and mexilitine, and use of opioid drugs in extreme cases.

Paroxysmal Extreme Pain Disorder is another rare disorder characterized by severe episodic pain in rectal, ocular, and mandibular regions as well as skin redness. Symptoms of this condition often begin in the neonatal period or in the early childhood, and can retain throughout life. Agents for treating chronic neuropathic pain disorders are often used to alleviate the pain episodes caused by the disease. Carbamazepine, a sodium channel blocker, has proven most effective of these treatments.

Mutations in the SCN10A gene are also known to cause pain perception disorders, including Familial Episodic Pain Syndrome Type 2 and Small Fiber Neuropathy. Thus, knock-out or knock-down of the SCN10A gene in patients having Familial Episodic Pain Syndrome Type 2 or Small Fiber Neuropathy can be used to treat, prevent and/or mitigate the associated symptoms.

Familial Episodic Pain Syndrome Type 2 is a rare autosomal dominant neurologic disorder characterized by adult-onset of paroxysmal pain in the feet region. The episodes are generally triggered by heat, cold, chemicals and certain surfaces. Patients may also develop hypersensitivity to touch and elevated response to pain stimulus. Currently no treatment is available for this disease. Warmth has been shown to relieve the pain episodes.

Small Fiber Neuropathy is a condition characterized by severe pain attacks and insensitivity to pain. The pain attacks are usually described as numbness, stabbing or burning, or abnormal skin sensations such as tingling or itchiness. Currently, there is no cure for small fiber peripheral neuropathy. Treatment options include intravenous immunoglobulin (IVIG) and plasmapheresis.

As described herein, indel rates and indel patterns were determined for gene editing systems comprising pairs of RNA-guided endonuclease (e.g., SpCas9 or SaCas9) and specific guide RNAs. The gene-editing systems described herein rely on the identification of specific pairs of effective RNA-guided endonuclease and guide RNAs pairs (e.g., those disclosed herein) that facilitate effective modification of a voltage-gated sodium channel gene, such as SCN9A or SCN10A, with low off target occurrence.

Accordingly, provided herein are gene-editing systems for efficient modification of voltage-gated sodium channel genes and uses thereof. Components of the gene-editing systems and genetically modified cells resulting from application of the gene-editing systems are also within the scope of the present disclosure.

I. Gene-Editing Systems for Genetic Modification of a Voltage-Gated Sodium Channel Gene

In some aspects, the disclosure relates to gene-editing systems for modifying a voltage-gated sodium channel gene, such as sodium voltage-gated channel alpha subunit 9 (SCN9A) or sodium voltage-gated channel alpha subunit 10 (SCN10A). A “gene-editing system” refers to a combination of components for editing a target gene (e.g., SCN9A or SCN10A), or one or more agents for producing such components. For example, a gene-editing system may comprise: (a) a nuclease, or an agent for producing such (e.g., a nucleic acid encoding the nuclease); and/or (b) a guide RNA (gRNA), or an agent for producing such (e.g., a vector capable of expressing the gRNA).

The gene-editing systems as described herein may exhibit one or more advantageous in modifying a voltage-gated sodium channel gene, such as SCN9A or SCN10A. For example, it would achieve a high gene editing rate, such as frameshift-causing indel rates (e.g., at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 30%, at least 35%, or at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% as assessed by methods described herein or known in the art) or such as total indel rates (e.g., at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 30%, at least 35%, or at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% as assessed by methods described herein or known in the art). Further, cells edited by the gene-editing system disclosed herein may have a high survival rate (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 85%, at least 90%, at least 95%, or at least 99%) relative to an unedited control.

In one exemplary embodiment, a gene-editing system as described herein may comprise: (a) an endonuclease (e.g., a RNA-guided DNA endonuclease) or an agent producing such (e.g., a polynucleotide coding for the endonuclease); and (b) a gRNA or an agent producing such (e.g., a vector for expressing the gRNA). Moreover, any of the gene editing systems described herein may further comprise a polynucleotide sequence encoding a donor template. In some examples, the gene-editing system described herein comprises an endonuclease, a gRNA, and optionally a donor template. Such a gene-editing system may comprise one polynucleotide that provides the donor template and produces the gRNA. Alternatively, the gene-editing system may comprise the donor template and a separate nucleic acid, which can be the gRNA per se, or a polynucleotide that produces the gRNA. In other examples, the gene-editing system may comprise one or more polynucleotides, which collectively produces the endonuclease, the gRNA, and optionally the donor template. In some examples, the gene-editing system may comprise a polynucleotide comprising a first polynucleotide sequence encoding an endonuclease and a second polynucleotide sequence encoding a gRNA. Alternatively, the gene-editing system may comprise two polynucleotides: the first comprising a first polynucleotide sequence encoding an endonuclease and the second comprising a second polynucleotide sequence encoding a gRNA.

A. RNA-Guided Endonucleases

RNA-guided endonucleases are enzymes that utilize RNA:DNA base-pairing to target and cleave a polynucleotide. RNA-guided endonuclease may cleave single-stranded polynucleic acids or at least one strand of a double-stranded polynucleotide. A gene editing-system may comprise one RNA-guided endonuclease. Alternatively, a gene-editing system may comprise at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more than ten) RNA-guided endonucleases.

The CRISPR-Cas9 system is a naturally-occurring defense mechanism in prokaryotes that has been repurposed as a RNA-guided DNA-targeting platform used for gene editing. It relies on the DNA nuclease Cas9, and two noncoding RNAs—crisprRNA (crRNA) and trans-activating RNA (tracrRNA)—to target the cleavage of DNA. crRNA drives sequence recognition and specificity of the CRISPR-Cas9 complex through Watson-Crick base pairing typically with a 20 nucleotide (nt) sequence in the target DNA. Changing the sequence of the 5′ 20 nt in the crRNA allows targeting of the CRISPR-Cas9 complex to specific loci. The CRISPR-Cas9 complex only binds DNA sequences that contain a sequence match to the first 20 nt of the crRNA if the target sequence is followed by a specific short DNA motif (with the sequence NGG) referred to as a protospacer adjacent motif (PAM). TracrRNA hybridizes with the 3′ end of crRNA to form a RNA-duplex structure that is bound by the Cas9 endonuclease to form the catalytically active CRISPR-Cas9 complex, which can then cleave the target DNA.

Once the CRISPR-Cas9 complex is bound to DNA at a target site, two independent nuclease domains within the Cas9 enzyme each cleave one of the DNA strands upstream of the PAM site, leaving a double-strand break (DSB) where both strands of the DNA terminate in a base pair (a blunt end).

A gene-editing system may comprise a CRISPR endonuclease (e.g., a CRISPR associated protein 9 or Cas9 nuclease). In some embodiments, the endonuclease is from Streptococcus aureus (e.g., saCas9) or Streptococcus pyogenes (e.g., spCas9), although other CRISPR homologs may be used. It should be understood that a Cas9 may be substituted with another RNA-guided endonuclease known in the art, such as Cpf1. Finally, it should be understood, that a wild-type RNA-guided endonuclease may be used or modified versions may be used (e.g., evolved versions of Cas9, Cas9 orthologues, Cas9 chimeric/fusion proteins, or other Cas9 functional variants). For example, in some embodiments, the RNA-guided endonuclease is modified to comprise a nuclear localization signal (NLS), such as an SV40 NLS or a NucleoPlasmine NLS. Examples of other nuclear localization signals are known to those having skill in the art. In some embodiments, the NLS comprises an SV40 NLS and a NucleoPlasmine NLS.

B. Guide RNA

The present disclosure provides a genome-targeting nucleic acid, or an agent for producing such (e.g., a polynucleotide comprising a nucleotide sequence encoding a gRNA), that can direct the activities of an associated polypeptide (e.g., a RNA-guided endonuclease) to a specific target sequence within a target nucleic acid. The genome-targeting nucleic acid can be a RNA. A genome-targeting RNA is referred to as a “guide RNA” or “gRNA” herein. In some embodiments, a gene-editing system comprises one gRNA. In other embodiments, a gene-editing system comprises at least two gRNAs (e.g., two, three, four, five, six, seven, eight, nine, ten, or more than ten gRNAs).

A gRNA of a gene-editing system may be provided in a synthesized form. For example, a guide RNA may be synthesized by chemical means, as illustrated below and described in the art. While chemical synthetic procedures are continually expanding, purifications of such RNAs by procedures such as high performance liquid chromatography (which avoids the use of gels such as PAGE) tends to become more challenging as polynucleotide lengths increase significantly beyond a hundred or so nucleotides. One approach used for generating RNAs of greater length is to produce two or more molecules that are ligated together. Much longer RNAs are more readily generated enzymatically. Various types of RNA modifications can be introduced during or after chemical synthesis and/or enzymatic generation of RNAs, e.g., modifications that enhance stability, reduce the likelihood or degree of innate immune response, and/or enhance other attributes, as described in the art.

Alternatively, a gene-editing system may comprise an agent for the production of a gRNA. For example, a gene-editing system may comprise a nucleotide sequence encoding the nucleotide sequence of a gRNA and an additional nucleotide sequence that facilitates expression/production of the gRNA.

A gRNA may be a double-molecule guide RNA. A double-molecule gRNA comprises two strands of RNA. The first strand may comprise in the 5′ to 3′ direction, an optional spacer extension sequence, a spacer sequence and a scaffold sequence comprising a minimum CRISPR repeat sequence. The second strand comprises a minimum tracrRNA sequence (complementary to the minimum CRISPR repeat sequence), a 3′ tracrRNA sequence, and an optional tracrRNA extension sequence.

Alternatively, a gRNA may be a single-molecule guide RNA (sgRNA) comprising a spacer sequence and a scaffold sequence. The scaffold sequence may comprise a tracrRNA sequence as described herein. A sgRNA (e.g., in a Type II system) may comprise, in the 5′ to 3′ direction, an optional spacer extension sequence, a spacer sequence, a minimum CRISPR repeat sequence, a single-molecule guide linker, a minimum tracrRNA sequence, a 3′ tracrRNA sequence and an optional tracrRNA extension sequence. The optional tracrRNA extension may comprise elements that contribute additional functionality (e.g., stability) to the guide RNA. The single-molecule guide linker links the minimum CRISPR repeat and the minimum tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension comprises one or more hairpins. Alternatively, a sgRNA (e.g., in a Type V system) may comprises, in the 5′ to 3′ direction, a minimum CRISPR repeat sequence and a spacer sequence.

The single-molecule gRNA can comprise no uracil at the 3′ end of the gRNA sequence. Alternatively, the gRNA can comprise one or more uracil at the 3′ end of the gRNA sequence. For example, the gRNA can comprise 1 uracil (U) at the 3′ end of the gRNA sequence. The gRNA can comprise 2 uracil (UU) at the 3′ end of the gRNA sequence. The gRNA can comprise 3 uracil (UUU) at the 3′ end of the gRNA sequence. The gRNA can comprise 4 uracil (UUUU) at the 3′ end of the gRNA sequence. The gRNA can comprise 5 uracil (UUUUU) at the 3′ end of the gRNA sequence. The gRNA can comprise 6 uracil (UUUUUU) at the 3′ end of the gRNA sequence. The gRNA can comprise 7 uracil (UUUUUUU) at the 3′ end of the gRNA sequence. The gRNA can comprise 8 uracil (UUUUUUUU) at the 3′ end of the gRNA sequence.

It is further understood that the nucleotides of the gRNAs described above may comprise modified nucleic acids at any nucleotide position. Accordingly, a gRNA can be unmodified or modified. For example, modified gRNAs can comprise one or more 2′-O-methyl phosphorothioate nucleotides. Examples of additional modified nucleic acids are known to those having skill in the art. See, e.g., WO2018007976 and WO2018007980, the relevant disclosures of each of which are incorporated by reference for the purpose and/or subject matter referenced herein.

(i) gRNA Spacer

As is understood by the person of ordinary skill in the art, each gRNA is designed to include a spacer sequence complementary to its genomic target sequence. See Jinek et al., Science, 337, 816-821 (2012) and Deltcheva et al., Nature, 471, 602-607 (2011). A spacer sequence is a nucleotide sequence that defines the target sequence (e.g., a DNA target sequences, such as a genomic target sequence) of a target nucleic acid of interest. The gRNA can comprise a variable length spacer sequence with 17-30 nucleotides at the 5′ end of the gRNA sequence. In some embodiments, the spacer sequence is 15 to 30 nucleotides. In some embodiments, the spacer sequence is 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, a spacer sequence is 20 nucleotides.

The “target sequence” is adjacent to a PAM sequence and is the sequence modified by a RNA-guided nuclease (e.g., Cas9). The “target nucleic acid” is a double-stranded molecule: one strand comprises the target sequence and is referred to as the “PAM strand,” and the other complementary strand is referred to as the “non-PAM strand.” One of skill in the art recognizes that the gRNA spacer sequence hybridizes to the reverse complement of the target sequence, which is located in the non-PAM strand of the target nucleic acid of interest. Thus, the gRNA spacer sequence is the RNA equivalent of the target sequence. For example, if the target sequence is 5′-AGAGCAACAGTGCTGTGGCC-3′ (SEQ ID NO: 498), then the gRNA spacer sequence is 5′-AGAGCAACAGUGCUGUGGCC-3′ (SEQ ID NO: 499). The spacer of a gRNA interacts with a target nucleic acid of interest in a sequence-specific manner via hybridization (i.e., base pairing). The nucleotide sequence of the spacer thus varies depending on the target sequence of the target nucleic acid of interest.

The spacer sequence is designed to hybridize to a region of the target nucleic acid that is located 5′ of a PAM of the Cas9 enzyme used in the system. The spacer may perfectly match the target sequence or may have mismatches. Each Cas9 enzyme has a particular PAM sequence that it recognizes in a target DNA. For example, S. pyogenes Cas9 recognizes in a target nucleic acid a PAM that comprises the sequence 5′-NRG-3′, where R comprises either A or G, where N is any nucleotide and N is immediately 3′ of the target nucleic acid sequence targeted by the spacer sequence. The canonical PAM for S. pyogenes Cas9 is 5′-NGG-3′, but as indicated in the preceding sentence, S. pyogenes Cas9 can also recognize the non-canonical PAM 5′-NAG-3′. Similarly, for S. aureus Cas9 the PAM comprises the sequence 5′-NNGRRT-3′.

In some embodiments, the target nucleic acid sequence comprises 20-22 nucleotides. In some embodiments, the target nucleic acid comprises less than 20 nucleotides. In some embodiments, the target nucleic acid comprises more than 20 nucleotides. In some embodiments, the target nucleic acid comprises at least: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. In some embodiments, the target nucleic acid comprises at most: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. In some embodiments, the target nucleic acid sequence comprises 20-22 bases immediately 5′ of the first nucleotide of the PAM. For example, in a sequence comprising 5′-NNNNNNNNNNNNNNNNNNNNNRG-3′ (SEQ ID NO: 489) or 5′-NNNNNNNNNNNNNNNNNNNNNNNNGRRT-3′ (SEQ ID NO: 490), the target nucleic acid comprises the sequence that corresponds to the Ns lacking an underscore, wherein N is any nucleotide, and the underlined NRG sequence and NNGRRT sequence is the S. pyogenes PAM and the S. aureus PAM, respectively.

In some embodiments, a gRNA used herein may comprise a spacer sequence of 20 nucleotides. In some embodiments, such a gRNA is used with a SpCas9. In other embodiments, a gRNA used herein may comprise a spacer sequence of 22 nucleotides. In some embodiments, such a gRNA is used with a SaCas9.

In some embodiments, a gRNA used herein may comprise a spacer sequence listed in Tables 1-4. In some examples, a gRNA used herein may comprise a spacer sequence listed in Table 1 in combination with SpCas9 for editing SCN9A. In some examples, a gRNA used herein may comprise a spacer sequence listed in Table 2 in combination with SaCas9 for editing SCN9A. In some examples, a gRNA used herein may comprise a spacer sequence listed in Table 3 in combination with SpCas9 for editing SCN10A. In some examples, a gRNA used herein may comprise a spacer sequence listed in Table 4 in combination with SaCas9 for editing SCN10A. Any of these gRNAs may comprise a spacer sequence listed in any of Tables 1 and 3 (in combination with SpCas9 enzyme) with greater than 40% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, or greater) mean total Indel percentage and/or with greater than 40% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, or greater) mean frameshift-causing Indel percentage. Alternatively, any of these gRNAs may comprise a spacer sequence listed in any of Tables 2 and 4 (in combination with SaCas9 enzyme) with greater than 15% (e.g., 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or greater) mean total Indel percentage and/or with greater than 15% (e.g., 20%, 25%, 30%, 35%, 40%, 45%, or greater) mean frameshift-causing Indel percentage.

Exemplary gRNAs may comprise one of the following spacer sequences:

(SEQ ID NO: 1) CAAUUUGGGUGGUACCUGAU; (SEQ ID NO: 2) GCUUCGCCUUGCAGAAAACA; (SEQ ID NO: 3) GCCUAUGCCCUUCGACACCA; (SEQ ID NO: 4) AUAGGCGAGCACAUGAAAAG; (SEQ ID NO: 5) CGGCUGAAUAUACAAGUAUU; (SEQ ID NO: 6) GGAACACCACCCAAUGACUG; (SEQ ID NO: 7) CAGGCCUGAAGACAAUUGUA; (SEQ ID NO: 8) GGAAUGUCCCCAUAGAUGAA; (SEQ ID NO: 9) CCACCAAUGCUGCCGGUGAA; (SEQ ID NO: 10) CAGUCACCACUCAGCAUUCG; (SEQ ID NO: 11) AAGCAGAAUUAUGGGCCUCUCA; (SEQ ID NO: 12) GCCUUGCAGAAAACAAGGAGCC; (SEQ ID NO: 13) ACGACAAAAUCCAGCCAGUUCC; (SEQ ID NO: 14) CUGGGAAAACCUUUACCAACAG; (SEQ ID NO: 15) UCCCAACCUCAGACAGAGAGCA; (SEQ ID NO: 16) GAUGUUACUGCUGCGUCGCUCC; (SEQ ID NO: 17) CAUGAUCCUGACUGUGUUCUGU; (SEQ ID NO: 18) CUCGUGUGUAGUCAGUGUCCAG; (SEQ ID NO: 19) AAACUGAUUGCCAUGGAUCCAU; (SEQ ID NO: 20) AGAAAACAAGGAGCCACGAAUG; (SEQ ID NO: 21) GCUCCCCGAUCAGUUCUGCU; (SEQ ID NO: 22) UGUAGUCACCAUGGCGUAUG; (SEQ ID NO: 23) GGAAGCUCCGCAGCACAGAC; (SEQ ID NO: 24) UCCUUACAACCAGCGCAGGA; (SEQ ID NO: 25) ACUUCUGACCCCUUACUGUG; (SEQ ID NO: 26) GAGCUCCCAGCAGAACUGAU; (SEQ ID NO: 27) CCGAGACAUCGACAGCUCCA; (SEQ ID NO: 28) AUCCGUUCUACAGCACACAC; (SEQ ID NO: 29) UCACGUACCUGAGAGAUCCU; (SEQ ID NO: 30) CGCAGGUGCUAGCAGCACUA; (SEQ ID NO: 31) CCCUGGAGCUGUCGAUGUCUCG; (SEQ ID NO: 32) UAGAUCCGUUCUACAGCACACA; (SEQ ID NO: 33) AGUGAGAGGAAAGCCCAAGCAA; (SEQ ID NO: 34) ACCUUUCCGGGCCCAAAGGGCA; (SEQ ID NO: 35) CUUUGACUGCAUCAUCGUCACU; (SEQ ID NO: 36) CACUUCUUCUGGAAAUAAUAGU; (SEQ ID NO: 37) AUUUUAGCGUCAUUACCCUGGC; (SEQ ID NO: 38) AACAACUUCCGUCGCUUUACUC; (SEQ ID NO: 39) GCCGAGAUAUCUCACUCCCUGA; (SEQ ID NO: 40) UGGUGUUCAUCUUCUCCAUGCC.

(ii) gRNA Scaffold

In some embodiments, the gRNA further comprises a scaffold sequence. A scaffold sequence may comprise the sequence of a minimum CRISPR repeat sequence, a single-molecule guide linker, a minimum tracrRNA sequence, a 3′ tracrRNA sequence, and/or an optional tracrRNA extension sequence. Exemplary scaffold sequences for various CRISPR proteins are known to those of ordinary skill in the art.

Selection of a scaffold sequence may depend on the RNA-guided DNA endonuclease to be used in the gene editing system as used herein, e.g., SaCas9 or SpCas9, which is known to those skilled in the art. For example, if SpCas9 is to be used, a scaffold sequence recognizable by SpCas9 can be selected. Examples of SpCas9 scaffold sequences are known in the art. See, e.g., Zhang et al., Plant Mol Biol. 2018; 96(4): 445-456; www.addgene.org. One exemplary scaffold sequence in a single-molecule guide RNA may comprise the nucleotide sequence of

(SEQ ID NO: 42) GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGT CCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC;

Alternatively, if a SaCas9 endonuclease is to be used, a scaffold sequence recognizable by the SaCas9 can be selected. A scaffold sequence in a single-molecule guide RNA for SaCas9 may comprise the nucleic acid sequence of GUUUUAGUACUCUGGAAACAGAAUCUACUAAA ACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUU (SEQ ID NO: 41). A single-molecule guide RNA may further comprise an optional spacer extension.

It should be understood that because the nucleotide sequence encoding a gRNA can be either a DNA sequence or a RNA sequence, any of the uracils (U) in the sequences describing a gRNA may be replaced with a thymine (T). Likewise, any T (thymine) in a sequence referring to gRNAs would refer to U (or uracil) in the context of RNA molecules. Sequences containing T (thymine) herein would encompass both DNA molecules and RNA molecules (wherein T refers to U).

(iii) Exemplary RNA-Guided Endonuclease-gRNA Pairs

In some embodiments, the gene editing system relies on the identification of effective RNA-guided endonuclease, guide RNAs pairs (e.g., those disclosed herein) for effective modification of a voltage-gated sodium channel gene.

For example, a gene editing system for modifying a sodium voltage-gated channel alpha subunit 9 (SCN9A) gene may comprise a Staphylococcus pyogenes (SpCas9) and a gRNA comprising the nucleotide sequence of any one of SEQ ID NOs: 1-10. Alternatively or in addition, a gene editing system for modifying a sodium voltage-gated channel alpha subunit 9 (SCN9A) gene may comprises a Staphylococcus aureus (SaCas9) and a gRNA comprising the nucleotide sequence of any one of SEQ ID NOs: 11-20.

In another example, a gene editing system for modifying a sodium voltage-gated channel alpha subunit 10 (SCN10A) gene may comprise a SpCas9 and a gRNA comprising the nucleotide sequence of any one of SEQ ID NOs: 21-30. Alternatively or in addition, a gene editing system for modifying a sodium voltage-gated channel alpha subunit 10 (SCN10A) gene may comprise a SaCas9 and a gRNA comprising the nucleotide sequence of any one of SEQ ID NOs: 31-40.

(iv) Ribonucleoprotein Complexes

In some instances, the gene-editing system disclosed herein may comprise a ribonucleoprotein complex (RNP), in which a gRNA and a nuclease (e.g., as described above) form a complex. As used herein, the term “ribonucleoprotein” or “RNP” refers to a protein that is structurally associated with a nucleic acid (either DNA or RNA). For example, in some embodiments, a Cas9 RNA-guided endonuclease and a gRNA of a gene-editing system are in the form of an RNP.

C. Donor Template

A donor template comprises a nucleic acid sequence that is to be inserted into a target site in a DNA sequence (e.g., in an endogenous gene). A donor template of a gene-editing system may be provided in a synthesized form. Alternatively, a gene-editing system may comprise an agent (e.g., a nucleic acid such as a vector) for the production of a donor template. For example, a gene-editing system may comprise a nucleic acid (e.g., a vector) for producing the donor template.

A donor template may comprise one or more homologous arms to allow for efficient homology dependent recombination (HDR) at a genomic location of interest. The length of a homologous arm may vary. For example, a homologous arm may be at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, at least 950, or at least 1000 nucleotides in length. Likewise, a homologous arm may be 50 to 100, 50 to 200, 50 to 300, 50 to 400, 50 to 500, 50 to 600, 50 to 700, 50 to 800, 50 to 900, 50 to 1000, 100 to 200, 100 to 300, 100 to 400, 100 to 500, 100 to 600, 100 to 700, 100 to 800, 100 to 900, 100 to 1000, 200 to 300, 200 to 400, 200 to 500, 200 to 600, 200 to 700, 200 to 800, 200 to 900, 200 to 1000, 300 to 400, 300 to 500, 300 to 600, 300 to 700, 300 to 800, 300 to 900, 300 to 1000, 400 to 500, 400 to 600, 400 to 700, 400 to 800, 400 to 900, 400 to 1000, 500 to 600, 500 to 700, 500 to 800, 500 to 900, 500 to 1000, 600 to 700, 600 to 800, 600 to 900, 600 to 1000, 700 to 800, 700 to 900, 700 to 1000, 800 to 900, 800 to 1000, or 900 to 1000 nucleotides in length. In particular, a homologous arm may be 500 nucleotides in length.

For example, in some embodiments a donor template comprises a 5′ homologous arm (i.e., positioned upstream to the first nucleotide sequence) and a 3′ homologous arm (i.e., positioned downstream to the first nucleotide sequence), wherein the 5′ homologous arm comprises a nucleic acid sequence that is homologous to a region upstream to the genomic location of interest, and wherein the 3′ homologous arm comprises a nucleic acid sequence that is homologous to a region downstream to the genomic location of interest.

In other embodiments, the donor template may comprise a 5′ homologous arm and lack a 3′ homologous arm. In yet other embodiments, the donor template may comprise a 3′ homologous arm and lack a 5′ homologous arm.

Alternatively, a donor template may lack homologous arms. For example, in some instances, a donor template may be integrated by NHEJ-dependent end joining following cleavage at the target site.

A donor template may also comprise a polynucleotide sequence encoding a gene of interest, or a portion thereof (e.g., SCN9A, SCN10A, or a portion thereof). Alternatively or in addition, a donor template may comprise a polynucleotide sequence encoding a regulatory element (e.g., a regulatory element of SCN9A or SCN10A)

A donor template can be DNA or RNA, single-stranded and/or double-stranded, and can be introduced into a cell in linear or circular form. If introduced in linear form, the ends of the donor sequence can be protected (e.g., from exonucleolytic degradation) by methods known to those of skill in the art. For example, one or more dideoxynucleotide residues are added to the 3′ terminus of a linear molecule and/or self-complementary oligonucleotides are ligated to one or both ends. See, for example, Chang et al., (1987) Proc. Natl. Acad. Sci. USA 84:4959-4963; Nehls et al., (1996) Science 272:886-889. Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group(s) and the use of modified internucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues.

A donor template can be introduced into a cell as part of a vector molecule having additional sequences such as, for example, replication origins, promoters and genes encoding antibiotic resistance. Moreover, a donor template can be introduced as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or poloxamer, or can be delivered by viruses (e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus and integrase defective lentivirus (IDLV)).

A donor template, in some embodiments, is inserted so that its expression is driven by the endogenous promoter, such as the promoter that drives expression of the endogenous gene into which the donor is inserted.

Furthermore, exogenous sequences may also include transcriptional or translational regulatory sequences, for example, promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding 2A peptides and/or polyadenylation signals.

It is understood that the nucleotides of the donor templates described above may comprise modified nucleic acids at any nucleotide position.

D. Viral Vector/Viral Particle-Based Gene-Editing System

In some embodiments, the gene-editing system disclosed herein may comprise polynucleic acids (e.g., vectors such as viral vectors) or viral particles comprising such. The polynucleic acid(s) produces the components (e.g., a nuclease and a gRNA) for editing a voltage-gated sodium channel gene as described herein.

In some examples, the gene-editing system comprises one polynucleic acid capable of producing all components of the gene-editing system, including a nuclease and a gRNA. In other examples, the gene-editing system comprises two polynucleic acids, one encoding the nuclease and the other encoding the gRNA.

The nucleic acid (or at least one nucleic acid in the set of nucleic acids) may be a vector such as a viral vector, such as a retroviral vector, an adenovirus vector, an adeno-associated viral (AAV) vector, and a herpes simplex virus (HSV) vector.

In some examples, the gene-editing system may comprise one or more viral particles that carry genetic materials for producing the components of the gene-editing system as disclosed herein. A viral particle (e.g., AAV particle) may comprise one or more components (or agents for producing one or more components) of a gene-editing system (e.g., as described herein). A viral particle (or virion) comprises a nucleic acid, which encodes the viral genome, and an outer shell of protein (i.e., a capsid). In some instances, a viral particle further comprises an envelope of lipids that surround the protein shell.

In some examples, a viral particle comprises a polynucleic acid capable of producing all components of the gene-editing system, including a nuclease and a gRNA. In other examples, a viral particle comprises a polynucleic acid capable of producing one or more components of the gene-editing system. For example a viral particle may comprise a polynucleic acid capable of producing the nuclease. Alternatively, a viral particle may comprise a polynucleic acid capable of producing the gRNA.

The viral particles described herein may be derived from any viral particle known in the art including, but not limited to, a retroviral particle, an adenovirus particle, an adeno-associated viral (AAV) particle, or a herpes simplex virus (HSV) particle. In some embodiments, the viral particle is an AAV particle. In some embodiments, the AAV particle is an AAV1 particle.

In some embodiments, a set of viral particles comprises more than one gene-editing system. In some embodiments, each viral particle in the set of viral particles is an AAV particle. In other embodiments, a set of viral particles comprises more than one type of viral particle (e.g., a retroviral particle, an adenovirus particle, an adeno-associated viral (AAV) particle, or a herpes simplex virus (HSV) particle).

E. Additional Exemplary Gene-Editing Systems

In addition, the gene-editing system disclosed herein may comprise a nuclease (e.g., a Cas9 enzyme) as disclosed herein. Such a gene-editing system may further comprise the gRNA. The nuclease and the gRNA may form an RNP for delivery. Further, the gene-editing system may further comprise the gRNA and a polynucleic acid (e.g., a vector as those described herein) for producing the donor template. The nuclease and the gRNA may form an RNP complex. Alternatively, the gene-editing system may further comprise one or more polynucleic acids for producing the gRNA and the donor template.

Alternatively, the gene-editing system disclosed herein may comprise an agent for produce the nuclease, for example, an expression vector such as a viral vector as disclosed herein capable of expressing the nuclease. Such a gene-editing system may further comprise the gRNA or agents for producing such.

Any other format of the gene-editing system comprising the components as disclosed herein for modifying a voltage-gated sodium channel gene or agents producing such are within the scope of the present disclosure.

II. Methods of Editing a Voltage-Gated Sodium Channel Gene

In some aspects, the disclosure relates to methods of editing a voltage-gated sodium channel gene, such as sodium voltage-gated channel alpha subunit 9 (SCN9A) or sodium voltage-gated channel alpha subunit 10 (SCN10A), using any of the gene-editing systems disclosed herein. An editing event may introduce a mutation or correct a mutation in a sodium voltage-gated channel (e.g., SCN9A or SCN10A). One or more copies (i.e., alleles) of a gene (e.g., SCN9A or SCN10A) may be corrected and/or mutated.

A method of editing a voltage-gated sodium channel gene may comprise contacting a cell with: a gene-editing system as described herein; a viral particle or set of viral particles comprising a gene-editing system as described herein; and/or a nucleic acid or set of nucleic acids comprising a gene-editing system as described herein. These methods may be performed, for example, on one or more cells existing within a living subject (e.g., in vivo). Alternatively or in addition, these methods may be performed on one or more cells existing in culture (e.g., ex vivo). In some instances, a cell edited in culture is then administered to a subject (categorized herein as “cell-based therapy”).

A. Delivery Methods

The contacting of the cell (or subject) with the gene-editing system, viral particle or set of viral particles, and/or nucleic acid or set of nucleic acids may be performed via various delivery methods. For example, nucleases and/or gRNAs may be delivered using a vector system, including, but not limited to, plasmid vectors, DNA minicircles, retroviral vectors, lentiviral vectors, adenovirus vectors, poxvirus vectors; herpesvirus vectors and adeno-associated virus vectors, and combinations thereof.

Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids encoding nucleases and gRNAs in cells. Non-viral vector delivery systems include DNA plasmids, DNA minicircles, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell.

Methods of non-viral delivery of nucleic acids include, but are not limited to, electroporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, naked RNA, capped RNA, artificial virions, and agent-enhanced uptake of DNA. Sonoporation using, e.g., the Sonitron 2000 system (Rich-Mar) can also be used for delivery of nucleic acids.

Methods for delivery of proteins (e.g., RNA-guided endonucleases) include, but are not limited to, the use of cell-penetrating peptides and nanovehicles.

(i) Adeno-Associated Viral Delivery

One or more components of a gene editing system may be delivered to a cell using an adeno-associated virus (AAV). AAVs are small viruses which integrate site-specifically into the host genome and can therefore deliver a transgene. Inverted terminal repeats (ITRs) are present flanking the AAV genome and/or the transgene of interest and serve as origins of replication. Also present in the AAV genome are rep and cap proteins which, when transcribed, form capsids which encapsulate the AAV genome for delivery into target cells. Surface receptors on these capsids confer AAV serotype, which determines which target organs the capsids will primarily bind and thus what cells the AAV will most efficiently infect. There are twelve currently known human AAV serotypes. In some embodiments, the AAV is AAV serotype 6 (AAV6). In some embodiments, the AAV is AAV serotype 1 (AAV1).

Adeno-associated viruses are among the most frequently used viruses for gene therapy for several reasons. First, AAVs do not provoke an immune response upon administration to mammals, including humans. Second, AAVs are effectively delivered to target cells, particularly when consideration is given to selecting the appropriate AAV serotype. Finally, AAVs have the ability to infect both dividing and non-dividing cells because the genome can persist in the host cell without integration. This trait makes them an ideal candidate for gene therapy.

(ii) Homology-Directed Repair (HDR)

One or more components of a gene editing system may be inserted into the target genomic region of the edited cell by homology directed repair (HDR). Both strands of the DNA at the target genomic region are cut by a CRISPR Cas9 enzyme. HDR then occurs to repair the double-strand break (DSB) and insert the donor DNA. For this to occur correctly, the donor sequence is designed with flanking residues which are complementary to the sequence surrounding the DSB site in the target gene (hereinafter “homology arms”). These homology arms serve as the template for DSB repair and allow HDR to be an essentially error-free mechanism. The rate of homology directed repair (HDR) is a function of the distance between the mutation and the cut site so choosing overlapping or nearby target sites is important. Templates can include extra sequences flanked by the homologous regions or can contain a sequence that differs from the genomic sequence, thus allowing sequence editing.

(iii) Non-Homologous End Joining (NHEJ)

The NHEJ pathway may also produce, at very low frequency, inserts containing exons 11-27. Such repair should correct expression when the insert is in the sense strand orientation.

III. Therapeutic Applications

The gene-editing methods disclosed herein may be applied for treating a patient with pain. In some embodiments, provided herein are ex vivo cell-based therapy. In other embodiments, provided herein are in vivo gene therapy.

(i) Cells-Based Therapy

Genetically-edited cells may be produced using any of the methods described herein. In some embodiments, one or more gene edits within a population of edited cells results in a phenotype associated with changes in voltage-gated sodium channel functionality.

In some embodiments, genetically-edited cells of the present disclosure exhibit decreased voltage-gated sodium channel activity (e.g., decreased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) relative to the unedited control. For example, the levels of Na_(v)1.7 and/or Na_(v)1.8 activity may be decreased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% relative to control unedited cells. In some embodiments, the levels of Na_(v)1.7 and/or Na_(v)1.8 activity may be decreased by 5%-10%, 5%-20%, 5%-30%, 5%-40%, 5%-50%, 5%-60%, 5%-70%, 5%-80%, 5%-90%, 10%-20%, 10%-30%, 10%-40%, 10%-50%, 10%-60%, 10%-70%, 10%-80%, 10%-90%-20%-30%, 20%-40%, 20%-50%, 20%-60%, 20%-70%, 20%-80%, 20%-90%, 30%-40%, 30%-50%, 30%-60%, 30%-70%, 30%-80%, 30%-90%, 40%-50%, 40%-60%, 40%-70%, 40%-80%, 40%-90%, 50%-50%, 50%-70%, 50%-80%, or 50%-90%, relative to control T cells.

In other embodiments, genetically-edited cells of the present disclosure exhibit increased voltage-gated sodium channel activity (e.g., by at least 30%, 50%, 100%, 2-fold, 5-fold, or 10-fold) relative to the unedited control. For example, the levels of Na_(v)1.7 and/or Na_(v)1.8 activity may be increased by at least 30%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000% relative to control unedited cells. In some embodiments, the levels of Na_(v)1.7 and/or Na_(v)1.8 activity may be increased by 30%-50%, 30%-100%, 30%-200%, 30%-500%, 30%-1000%, 50%-100%, 50%-200%, 50%-500%, 50%-1000%, 100%-200%, 100%-500%, 100%-1000%, 200%-500%, 200%-1000%, or 500%-1000% relative to control unedited cells.

In some embodiments, a biopsy of the patient's peripheral nerves can be performed. The nerve tissue can be isolated from the patient's skin or leg. Then, a cell of the peripheral nervous system (e.g., a neuron or a glial cell such as Schwann cell in nerves or satellite glial cell in ganglia) is isolated from the biopsied material. Then, the chromosomal DNA of the cell of the peripheral nervous system (e.g., a neuron, or a glial cell such as Schwann cell in nerves or satellite glial cell in ganglia) can be edited using the materials and methods described herein. Finally, the edited cell of the peripheral nervous system (e.g., a neuron or a glial cell such as Schwann cell in nerves or satellite glial cell in ganglia) is implanted into the patient. Any source or type of cell may be used as the progenitor cell.

In other embodiments, a patient specific induced pluripotent stem cell (iPSC) can be created. Then, the chromosomal DNA of these iPSC cells can be edited using the materials and methods described herein. Next, the genome-edited iPSCs can be differentiated into cells of the peripheral nervous system (e.g., a neuron or a glial cell such as Schwann cell in nerves or satellite glial cell in ganglia). Finally, the differentiated cells of the peripheral nervous system (e.g., a neuron or a glial cell such as Schwann cell in nerves or satellite glial cell in ganglia) are implanted into the patient.

Alternatively, a mesenchymal stem cell can be isolated from the patient, which can be isolated from the patient's bone marrow or peripheral blood. Next, the chromosomal DNA of these mesenchymal stem cells can be edited using the materials and methods described herein. Next, the genome-edited mesenchymal stem cells can be differentiated into cells of the peripheral nervous system (e.g., a neuron or a glial cell such as Schwann cell in nerves or satellite glial cell in ganglia). Finally, the differentiated cells of the peripheral nervous system (e.g., a neuron or a glial cell such as Schwann cell in nerves or satellite glial cell in ganglia) are implanted into the patient.

Any of the genetically edited cells may be administered to a subject. The step of administering may include the placement (e.g., transplantation) of genetically engineered cells into a subject, by a method or route that results in at least partial localization of the introduced cells at a desired site, such that a desired effect(s) is produced and where at least a portion of the implanted cells or components of the cells remain viable. The period of viability of the cells after administration to a subject can be as short as a few hours, e.g., twenty-four hours, to a few days, to as long as several years, or even the life time of the subject, i.e., long-term engraftment. In some embodiments, the administration is to the respiratory tract of the subject.

Modes of administration include injection, infusion, instillation, or ingestion. Injection includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrasternal injection and infusion. In some embodiments, the route is intravenous.

In some embodiments, genetically engineered cells are administered systemically, which refers to the administration of a population of cells other than directly into a target site, tissue, or organ, such that it enters, instead, the subject's circulatory system and, thus, is subject to metabolism and other like processes.

For use in the various aspects described herein, an effective amount of genetically engineered cells comprises at least 10² cells, at least 5×10² cells, at least 10³ cells, at least 5×10³ cells, at least 10⁴ cells, at least 5×10⁴ cells, at least 10⁵ cells, at least 2×10⁵ cells, at least 3×10⁵ cells, at least 4×10⁵ cells, at least 5×10⁵ cells, at least 6×10⁵ cells, at least 7×10⁵ cells, at least 8×10⁵ cells, at least 9×10⁵ cells, at least 1×10⁶ cells, at least 2×10⁶ cells, at least 3×10⁶ cells, at least 4×10⁶ cells, at least 5×10⁶ cells, at least 6×10⁶ cells, at least 7×10⁶ cells, at least 8×10⁶ cells, at least 9×10⁶ cells, or multiples thereof. In some examples described herein, the cells are expanded in culture prior to administration to a subject in need thereof.

(ii) In Vivo Gene Therapy

Alternatively, the gene-editing methods and materials disclosed herein can be applied to genetically modifying the target gene (SCN9A or SCN10A) in vivo. Chromosomal DNA of the cells in a patient can be edited using the materials and methods described herein. In some aspects, the target cell in an in vivo based therapy can be a neuron of the peripheral nervous system.

Although certain cells present an attractive target for ex vivo treatment and therapy, increased efficacy in delivery may permit direct in vivo delivery to such cells. Ideally the targeting and editing would be directed to the relevant cells. Cleavage in other cells can also be prevented by targeted delivery and/or the use of promoters only active in certain cells and or developmental stages. Additional promoters are inducible, and therefore can be temporally controlled if the nuclease is delivered as a plasmid. The amount of time that delivered RNA and protein remain in the cell can also be adjusted using treatments or domains added to change the half-life. In vivo treatment would eliminate a number of treatment steps, but a lower rate of delivery can require higher rates of editing. In vivo treatment can eliminate problems and losses from ex vivo treatment and engraftment and post-engraftment integration of neurons and glial cells appropriately into existing brain circuits.

In some aspects, the disclosure relates to methods of administering an effective amount of a gene-editing system as descried herein, a viral particle or set of viral particles comprising a gene-editing system as described herein, a nucleic acid or set of nucleic acids comprising a gene-editing system as described herein, or a composition of edited cells as described herein to a subject in need thereof.

A subject may be any subject for whom diagnosis, treatment, or therapy is desired. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a human patient having pain. In some embodiments, the human patient is a child.

An effective amount refers to the amount of a gene-editing system, a viral particle or set of viral particles comprising a gene-editing system, a nucleic acid or set of nucleic acids comprising a gene-editing system, or a population of genetically engineered cells needed to prevent or alleviate at least one or more signs or symptoms of a medical condition (i.e., pain), and relates to a sufficient amount of a composition to provide the desired effect (i.e., to treat a subject having pain). An effective amount also includes an amount sufficient to prevent or delay the development of a symptom of the disease, alter the course of a symptom of the disease (for example but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of the disease. It is understood that for any given case, an appropriate effective amount can be determined by one of ordinary skill in the art using routine experimentation.

The efficacy of a treatment comprising a composition for the treatment of a medical condition can be determined by the skilled clinician. A treatment is considered an “effective treatment,” if any one or all of the signs or symptoms of, as but one example, levels of functional target are altered in a beneficial manner (e.g., increased by at least 10%), or other clinically accepted symptoms or markers of disease (e.g., pain) are improved or ameliorated. Efficacy can also be measured by failure of a subject to worsen as assessed by hospitalization or need for medical interventions (e.g., progression of the disease is halted or at least slowed). Methods of measuring these indicators are known to those of skill in the art and/or described herein. Treatment includes any treatment of a disease in subject and includes: (1) inhibiting the disease, e.g., arresting, or slowing the progression of symptoms; or (2) relieving the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of the development of symptoms.

IV. Kits for Therapeutic Use

The present disclosure also provides kits for use of the compositions described herein. For example, the present disclosure provides kits comprising a gene-editing system as described herein; a viral particle or set of viral particles comprising a gene-editing system as described herein; a nucleic acid or set of nucleic acids comprising a gene-editing system as described herein; and/or a population of genetically-edited cells as described herein.

In some embodiments, the kit can additionally comprise instructions for use in any of the methods described herein. The included instructions may comprise a description of: (i) the delivery of a gene-editing system as described herein; a viral particle or set of viral particles comprising a gene-editing system as described herein; and/or a nucleic acid or set of nucleic acids comprising a gene-editing system as described herein; and/or (ii) the administration of a population of genetically-edited cells as described herein.

The kit may further comprise a description of selecting a subject suitable for treatment based on identifying whether the subject is in need of the treatment. The instructions may include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the disclosure are typically written instructions on a label or package insert. The label or package insert indicates that the pharmaceutical compositions are used for treating, delaying the onset, and/or alleviating a disease or disorder in a subject.

The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. Also contemplated are packages for use in combination with a specific device, such as an inhaler, nasal administration device, or an infusion device. A kit may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port.

Kits optionally may provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiment, the disclosure provides articles of manufacture comprising contents of the kits described above.

General Techniques

The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed. 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1989) Academic Press; Animal Cell Culture (R. I. Freshney, ed. 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds. 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds. 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (D. N. Glover ed. 1985); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. (1985»; Transcription and Translation (B. D. Hames & S. J. Higgins, eds. (1984»; Animal Cell Culture (R. I. Freshney, ed. (1986»; Immobilized Cells and Enzymes (IRL Press, (1986»; and B. Perbal, A practical Guide To Molecular Cloning (1984); F. M. Ausubel et al. (eds.).

Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present disclosure to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.

EXAMPLES Example 1. Efficacy Screening of SpCas9 and SaCas9 gRNAs Targeted to SCN9A and SCN10A in iPSCs

Methods

Guide RNA Design and Synthesis

In silico guide RNA design was completed by CRISPR Therapeutics. SpCas9 and SaCas9 guide RNAs targeting exons 2-15 of SCN9A and Exons 1-14 of SCN10A were designed in silico and evaluated using an off-target prediction algorithm. Guide RNAs with a favorable off-target profile were selected for synthesis and further on target evaluation. Selected gRNAs included 99 SpCas9 gRNAs (Table 1) and 68 SaCas9 gRNAs targeting SCN9A (Table 2) and 166 SpCas9 gRNAs (Table 3) and 73 SaCas9 gRNAs targeting SCN10A (Table 4).

Guide RNAs were custom ordered for synthesis by Synthego Corporation. Guide RNAs were ordered with standard chemical modifications which include 2′-O-methyl 3′ phosphorothioate modifications in the first and last 3 nucleotides. For SpCas9 gRNAs, the 20-nucleotide genome targeting sequences are listed in Tables 1 and 3, and a standard 80-mer SpCas9 scaffold sequence was added to create a guide RNA. For SaCas9 gRNAs, 22-nucleotide genome targeting sequences are listed in Tables 2 and 4 and were used for synthesis with the following SaCas9 scaffold sequence to generate guide RNAs: GUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAA GGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUU (SEQ ID NO: 41).

Nucleofection of iPSCs using 4D-Nucleofector® System

Wildtype iPSCs, as well as engineered iPSCs stably expressing Cas9, were used for different steps of gRNA screening. iPSCs expressing SpCas9 or SaCas9 under the control of doxycycline were generated from wildtype iPSCs by inserting a targeting construct into the AAVS-1 locus. In this construct, two cassettes are expressed in opposing directions separated by an IS2 insulator element. The first expression cassette is a TetOn3G protein-2A-Puro under the control of the CASI promoter, and the second expression cassette is either SpCas9 or SaCas9 under the control of the TRE3G promoter.

iPSCs were electroporated using the Lonza 4D-Nucleofector® System together with the P3 Primary Cell 96-well Nucleofector™ Kit (Lonza, Cat: V4SP-3096) with program CM137. iPSCs were cultured in mTeSR1 (Stemcell Technologies, Cat: 85850). Prior to nucleofection cells were dissociated using Accutase (Stemcell Technologies, Cat: 07920) and resuspended in P3 Nucleofection solution. In 96-well format, 180,000 cells per well were electroporated with 400 ng Cas9 mRNA (TriLink) per well and 400 ng synthetic gRNA (Synthego) according to manufacturer's instructions. Following nucleofection iPSCs were maintained in mTeSR1 supplemented with 10 uM Y27632 (Stemcell Technologies, Cat: 72308) in 96 well cell culture plates pre-coated with matrigel for 72 hours prior to DNA extraction and next generation sequencing (NGS)-based insertion/deletion (Indel) detection. Two replicates were included in each electroporation experiment and two independent experiments were carried out. For stable SpCas9 and SaCas9 cell line experiments, cells were treated with 1 ug/ul doxycycline for 72 hours prior to Amaxa nucleofection.

Nucleofection of iPSC-Derived Sensory Neurons Using Lonza 4D-Nucleofector® Y Unit

To generate iPSC-derived sensory neuron cultures (iSNs), iPSC cells were differentiated in the presence of a cocktail of small molecule developmental pathway inhibitors in matrigel coated flasks. At DIV11 of differentiation, cells were dissociated and plated into 384 plates and maintained in maturation media which includes a cocktail of growth factors where they matured until DIV26-28. These neurons express canonical markers of nociceptors, including TRPV1, Brn3A, the peripheral marker Isl1, neuN and SCN9A (NaV1.7), and can recapitulate functional properties of physiologically relevant neuronal subtypes.

iPSC-derived sensory neurons (iSNs) were electroporated using the Lonza 4D-Nucleofector® Y Unit together with the AD1 4D-Nucleofector™ Y Kit (Lonza, Cat: V4YP-1A24) with program EH158. In 24-well format, cells were electroporated with ribonucleoprotein complexes (RNPs) according to manufacturer's instructions. RNP complexes were generated by incubating 425 pmol SpCas9 or SaCas9 protein (Aldevron) with 531 pmol synthetic gRNA (Synthego) at room temperature for 20 minutes. Following nucleofection iSNs were maintained in culture for 72 hours prior to DNA extraction and next generation sequencing (NGS)-based insertion/deletion (Indel) detection. Two replicates were included in each electroporation experiment and two independent experiments were carried out.

Transduction of iPSC-Derived Sensory Neurons with AAV

In 384-well format, approximately 12,000 iSNs per well were transduced with AAV-1 vectors expressing SaCas9 and a SaCas9 gRNA in a single vector. iSNs were transduced with AAV vectors at a multiplicity of infection (MOI) of 750,000. Following transduction, iSNs were maintained in culture for 7 days prior to DNA extraction and NGS based insertion/deletion (indel) detection. Two replicates were included in each transduction experiment and two independent experiments were carried out.

Next Generation Sequencing (NGS) Based Insertion/Deletion (Indel) Detection

DNA was extracted from iPSCs 72 hours post electroporation using Lucigen Quick Extract 2×DNA Extraction Solution (Lucigen, Cat: QE09050) according to manufacturer's instructions. A two-step PCR approach using KAPA2G Robust HotStart ReadyMix (Sigma Aldrich, Cat: KK5702) was then used to generate NGS libraries. The first PCR was used to create amplicons while the second PCR was used to add Nextera DNA Index (i7/i5) adapter sequences. The reaction for PCR #1 comprised of 1 uL extracted gDNA, 1×KAPA2G Robust HotStart ReadyMix, 0.5 uM forward primer, and 0.5 uM reverse primer. Primer sequences are listed in Tables 5 and 6. The reaction for PCR #2 comprised of 1 ul PCR #1 product, 1×KAPA2G Robust HotStart ReadyMix, 0.5 uM Index 1 N7xx adapter, and 0.5 uM Index 2 N5xx adapter. Cycling conditions for both PCR #1 and PCR #2 were as follows: (1) 95° C. for 3 min, (2) 95° C. for 15 s, (3) 60° C. for 15 s, (4) 72° C. for 15 s, (5) repeat steps (2)-(4) 20 times, (6) 72° C. for 1 min, (7) 4° C. infinite hold. Samples were then pooled and purified using the Zymo DNA Clean and Concentrator Kit (Zymo, D4034) and quantified on the Agilent 2100 Bioanalyzer (Agilent, Cat: G2939BA). Then, libraries were run on Illumina's MiSeq to obtain paired-end reads (2×150).

For each sample, the reads were then filtered to obtain a minimum Phred33 quality score of 30. Paired-end reads were subsequently merged using FLASH (Fast Length Adjustment of SHort reads) with a requirement of at least 1 bp overlap. The resulting merged reads were then optimally aligned to the corresponding reference amplicon sequences using the Needleman-Wunsch algorithm. Reads that aligned with indels within 3 bp of the expected cut site were counted, and then filtered for frame-shifting indels only, where the indel length is not a multiple of 3. An estimate of total editing was calculated as the proportion of reads with indels proximal to the cut site, while productive editing was calculated as the proportion of reads with frame-shifting indel reads proximal to the cut site for each sample.

Once each sample was analyzed, quality control of the samples was then performed by requiring each sample to have at least 90% of sequenced reads successfully merged, and 70% of sequenced reads successfully aligned. Additionally, samples were required to have at least 1000 reads successfully aligned. Lastly, quality control was performed in a batch-aware manner, by dropping any samples whose final aligned read count was more than 2 standard deviations away from the mean of its corresponding batch of samples. Passing samples were averaged with the standard deviation calculated. Positive and negative controls were included, where the negative control was required to exhibited less than 2% indel rates, indicating a low level of background noise, and while the positive control samples had to show levels of editing above background. Further, reproducibility was confirmed by comparing corresponding samples between our two technical replicates; a strong linear fit was observed with a high R² of 0.85.

Off-Target Evaluation of SpCas9 gRNAs Targeted to SCN9A and SCN10A in iPSCs

For an initial off-target evaluation, an in silico nomination step was performed where candidate off-target sequences were predicted based on sequence similarity. Then, these sites were directly evaluated via targeted Next-Generation Sequencing to identify which sites, if any, showed evidence of CRISPR-Cas-induced off-target editing.

a) Computational Prediction of Off-Target Sites

Off-target sites were predicted based on sequence similarity using three computational algorithms. Specifically, CCTop and COSMID were each used to identify candidate off-target sites with up to 3 mismatches or up to 2 mismatches with 1 DNA or RNA bulge from the on-target sequence. The PAM sequence used for identifying off-target sites was NRG for SpCas9 guides and NNGRRT for SaCas9 guides. Guides identified from the two algorithms were then merged together, including de-duplication of sites with identical genomic coordinates. The total list of 1,471 putative off-target sites predicted across the 40 guides is provided in Table 7.

b) Hybrid Capture of iPSCs

iPSC transfections using two different wildtype donors were performed using the Lonza conditions described above. Two biological replicates were used, and genomic DNA pooled to obtain the necessary amount for hybrid capture. DNA was extracted from iPSCs 72 hours post electroporation using the DNeasy 96 Blood and Tissue Kit (Qiagen, Cat: 69581). Samples were quantified using the Qubit 1×dsDNA HS Assay (ThermoFisher, Cat: Q33231) and EnVision plate reader with 4PL calculation. A minimum of 200 ng of each sample was obtained and processed for hybrid capture using the SureSelect XT Reagent Kit (Aglient, Cat: G9704A). Briefly, samples were fragmented to 150-200 bp using the Covaris LE220 and end repaired, dA-tailed, and adapter ligated. Libraries were then amplified using Herculase II Fusion DNA polymerase using the following cycle conditions: (1) 98° C. for 2 min, (2) 98° C. for 30 s, (3) 65° C. for 30 s, (4) 72° C. for 1 min, (5) repeat steps (2)-(4) 10 times, (6) 72° C. for 5 min, (7) 4° C. infinite hold. A bead-based clean-up was used to purify libraries, AMPure XP (Beckman Coulter, Cat: A63881). Libraries were hybridized to the target-specific Capture Library and target molecules captures with Steptavidin-coated magnetic beads. Captured libraries were then amplified and purified using the same conditions above. Samples were QC'd using the DNA High Sensitivity kits on the TapeStation (Agilent, Cat: 5067-5584) and/or Bioanalyer (Aglient, Cat: 5067-1504) and sequenced on the Illumina HiSeq platform to a median sequencing coverage of 2,272× per candidate off-target site.

c) Computational Analysis of Targeted Next-Generation Sequencing

For each putative off-target site included in this study, the following analysis was performed to determine strength of evidence for CRISPR-Cas-treatment-induced off-target editing. First, next-generation sequencing reads were aligned to the hg38 human reference genome using the alignment tool bwa in the mem mode with default parameters, followed by conversion and sorting of the SAM and BAM files with read duplicate removal performed by samtools. Then, the indel formation rate was measured by piling up reads with an indel within 3 bp of the expected cut site using the Python package pysam and dividing the number of indel reads by the total number of reads covering that site.

Then, the indel rate measured at each predicted off-target site was compared between the treated sample of each iPSC donor and the untreated (electroporated only) negative control sample matched for that same iPSC donor. If an indel rate at the site was observed to be >0.2% greater than the negative control sample, the data for that candidate site entered statistical testing. The only exception was for candidate sites that were observed to have a germline indel genetic variant, where an indel rate of ˜50% or ˜100% is observed in both the matched untreated and treated sample of one or more donors. For sites that entered statistical testing, a paired t-test was performed across both donors for the treated and untreated indel rates at that site. Any tested site that resulted in a p-value of less than 0.05 was considered to have confirmed off-target editing. Substantial on-target editing (average indel rate of 9.35%-52.25%) was confirmed across guides as a positive control for this study.

TABLE 1  Names and sequences of SpCas9 guide RNAs targeted to the SCN9A gene (Nav1.7) Nav1.7 SpCas9 gRNAs SEQ spacer sequence ID NO: gRNA Name (5′-3′) 43 Scn9a Sp Exon 2_T1 AuCuAuGGGGACAuuCCuCC 44 Scn9a Sp Exon 2_T2 CAGCAAuGCGuuGuuCAAuG 45 Scn9a Sp Exon 2_T3 AGuAGGGGuCCAAGuCCuCC 46 Scn9a Sp Exon 2_T4 uGGGGACAuuCCuCCCGGCA 47 Scn9a Sp Exon 2_T5 GuAGGGGuCCAAGuCCuCCA 48 Scn9a Sp Exon 2_T6 uAGGGGuCCAAGuCCuCCAG 49 Scn9a Sp Exon 2_T7 AuGGCAAuGuuGCCuCCCCC 50 Scn9a Sp Exon 2_T8 GGCuCuGACACCAuGCCGGG 51 Scn9a Sp Exon 2_T9 AACAGCuGCCCuuCAuCuAu 8 Scn9a Sp Exon 2_T10 GGAAuGuCCCCAuAGAuGAA 52 Scn9a Sp Exon 2_T11 CGGCAuGGuGuCAGAGCCCC 53 Scn9a Sp Exon 2_T12 AGCAAuGCGuuGuuCAAuGA 54 Scn9a Sp Exon 2_T13 AGGAAuGuCCCCAuAGAuGA 55 Scn9a Sp Exon 2_T14 AAACAGCuGCCCuuCAuCuA 56 Scn9a Sp Exon 2_T15 CCCCuACuAuGCAGACAAAA 57 Scn9a Sp Exon 4_T1 CCAuGAAuAACCCACCGGAC 58 Scn9a Sp Exon 4_T2 CAuuuuuGGuCCAGuCCGGu 59 Scn9a Sp Exon 4_T3 CCAGuCCGGuGGGuuAuuCA 60 Scn9a Sp Exon 4_T4 ACAuuuuuGGuCCAGuCCGG 61 Scn9a Sp Exon 4_T5 uCGACAuuuuuGGuCCAGuC 62 Scn9a Sp Exon 4_T6 ACCCACuuACuCGACAuuuu 63 Scn9a Sp Exon 4_T7 uAuGACCAuGAAuAACCCAC 64 Scn9a Sp Exon 5_T1 uuCuuCGuGACCCGuGGAAC 65 Scn9a Sp Exon 5_T2 uCGuGACCCGuGGAACuGGC 66 Scn9a Sp Exon 5_T3 uCACuuuuCuuCGuGACCCG 67 Scn9a Sp Exon 5_T4 GuGuuuAGGuACACuuuuAC 68 Scn9a Sp Exon 7_T1 AAGCCCCuACAAuuGuCuuC 69 Scn9a Sp Exon 7_T2 CuGAGuGuGuuuGCACuAAu 70 Scn9a Sp Exon 7_T3 AuuGGACuACAGCuGuuCAu 7 Scn9a Sp Exon 7_T5 CAGGCCuGAAGACAAuuGuA 71 Scn9a Sp Exon 7_T6 AGGCCuGAAGACAAuuGuAG 72 Scn9a Sp Exon 9_T1 AAGCuCGuGuAGCCAuAAuC 73 Scn9a Sp Exon 9_T2 AGCuCGuGuAGCCAuAAuCA 74 Scn9a Sp Exon 9_T3 GGCuAAuGACCCAAGAuuAC 75 Scn9a Sp Exon 9_T4 uuCACACAGGuGuACCCCuC 76 Scn9a Sp Exon 9_T5 CGuGuGuAGuCAGuGuCCAG 77 Scn9a Sp Exon 9_T6 GCuAAuGACCCAAGAuuACu 78 Scn9a Sp Exon 9_T7 CGAGCuuuGACACuuuCAGC 79 Scn9a Sp Exon 9_T8 uGGuACuCACCuGuuGGuAA 80 Scn9a Sp Exon 9_T9 GGGuACACCuGuGuGAAAAu 81 Scn9a Sp Exon 9_T10 CuGGGAAAACCuuuACCAAC 82 Scn9a Sp Exon 9_T11 uuGGGuCAuuAGCCuAAACA 83 Scn9a Sp Exon 9_T12 GuGuGuAGuCAGuGuCCAGA 84 Scn9a Sp Exon 9_T13 GGGCCuuCuuAGCCuuGuuu 85 Scn9a Sp Exon 9_T14 GAGCuuuGACACuuuCAGCu 86 Scn9a Sp Exon 9_T15 AuuGGCAGAAACCCuGAuuA 87 Scn9a Sp Exon 10_T1 CCCuAGACGCuGCGuGCuGC 88 Scn9a Sp Exon 10_T2 CCAGCAGCACGCAGCGuCuA 89 Scn9a Sp Exon 10_T4 GCCAGCAGCACGCAGCGuCu 90 Scn9a Sp Exon 10_T5 CuuuGuCGuAGuGAuuuuCC 91 Scn9a Sp Exon 11_T2 GAGGuuGuCuACCCCCAAuC 92 Scn9a Sp Exon 11_T3 uAuGCCCuuCGACACCAAGG 93 Scn9a Sp Exon 11_T5 ACCuuGGuGuCGAAGGGCAu 3 Scn9a Sp Exon 11_T6 GCCuAuGCCCuuCGACACCA 94 Scn9a Sp Exon 11_T7 AGuuuCCACCuuGGuGuCGA 1 Scn9a Sp Exon 11_T9 CAAuuuGGGuGGuACCuGAu 95 Scn9a Sp Exon 11_T10 GuuuCCACCuuGGuGuCGAA 96 Scn9a Sp Exon 11_T11 CCGCuGCCGCuGCAAuuGCC 97 Scn9a Sp Exon 11_T12 GCCCAACCAGGCAAuuGCAG 5 Scn9a Sp Exon 11_T13 CGGCuGAAuAuACAAGuAuu 4 Scn9a Sp Exon 11_T14 AuAGGCGAGCACAuGAAAAG 98 Scn9a Sp Exon 11_T15 AAuuuGGGuGGuACCuGAuu 99 Scn9a Sp Exon 12_T1 CGuuCACCGGCAGCAuuGGu 100 Scn9a Sp Exon 12_T2 CCGuuCACCGGCAGCAuuGG 101 Scn9a Sp Exon 12_T3 uGuuACuGCuGCGuCGCuCC 102 Scn9a Sp Exon 12_T4 GuuACuGCuGCGuCGCuCCu 103 Scn9a Sp Exon 12_T5 GGGCuGAGCGuCCAuCAACC 104 Scn9a Sp Exon 12_T6 CACCAAuGCuGCCGGuGAAC 491 Scn9a Sp Exon 12_T7 uuCCCGuuCACCGGCAGCAu 105 Scn9a Sp Exon 12_T8 GGCuGAGCGuCCAuCAACCA 492 Scn9a Sp Exon 12_T9 GuuCACCGGCAGCAuuGGuG 106 Scn9a Sp Exon 12_T10 uuACuGCuGCGuCGCuCCuG 9 Scn9a Sp Exon 12_T11 CCACCAAuGCuGCCGGuGAA 107 Scn9a Sp Exon 12_T12 CuGCAACGGuGuGGuCuCCC 108 Scn9a Sp Exon 12_T13 CAGCAuuGGuGGGGACCuAC 493 Scn9a Sp Exon 12_T14 uuGGuGGGGACCuACuGGCu 109 Scn9a Sp Exon 12_T16 GCGuCGCuCCuGGGGuCuGu 10 Scn9a Sp Exon 12_T17 CAGuCACCACuCAGCAuuCG 494 Scn9a Sp Exon 12_T18 uAGGuCCCCACCAAuGCuGC 110 Scn9a Sp Exon 12_T19 uGCuGuGGACuGCAACGGuG 111 Scn9a Sp Exon 12_T20 CGuCGCuCCuGGGGuCuGuG 112 Scn9a Sp Exon 12_T21 uGCGuCGCuCCuGGGGuCuG 113 Scn9a Sp Exon 12_T22 GGuGuGGuCuCCCuGGuuGA 114 Scn9a Sp Exon 12_T23 GuAACAuCAGCCAAGCCAGu 115 Scn9a Sp Exon 12_T24 CuGuGCAuuuuCCCGuuCAC 2 Scn9a Sp Exon 12_T25 GCuuCGCCuuGCAGAAAACA 116 Scn9a Sp Exon 12_T26 GuuuGuGCCCCACAGACCCC 495 Scn9a Sp Exon 12_T27 GCuGuCCAuuGGGGAGCAuG 496 Scn9a Sp Exon 12_T28 uCuGGCAGAAGCuGuCCAuu 6 Scn9a Sp Exon 14_T1 GGAACACCACCCAAuGACuG 117 Scn9a Sp Exon 14_T2 GCAAAuCuGuACCACCAAGG 118 Scn9a Sp Exon 14_T3 uGuGCAAAuCuGuACCACCA 119 Scn9a Sp Exon 14_T4 CCAAGGuGGACAuuuuuGuC 120 Scn9a Sp Exon 14_T5 uGuCuGGACuCuuCAAGuuC 121 Scn9a Sp Exon 14_T6 uCuGGAAuuGCuCuCCAuAu 122 Scn9a Sp Exon 15_T1 AGuuCuGCGAuCAuuCAGAC 123 Scn9a Sp Exon 15_T2 GGAGCuCuuuCuAGCAGAuG 124 Scn9a Sp Exon 15_T3 CCuACuuGGAAAuACuCAuA

TABLE 2  Names and sequences of SaCas9 guide RNAs targeted to the SCN9A gene (Nav1.7) Nav1.7 SaCas9 gRNAs SEQ ID NO: gRNA Name spacer sequence (5′-3′) 125 Scn9a Sa Exon 2_T1 GGGGCuCuGACACCAuGCCGGG 126 Scn9a Sa Exon 2_T2 CCCCuACuAuGCAGACAAAAAG 127 Scn9a Sa Exon 2_T3 CuCACCuuuuuGuCuGCAuAGu 128 Scn9a Sa Exon 2_T4 AuCAuCAuCuuuCuuuuCuuCu 129 Scn9a Sa Exon 3_T1 uuuCuCCuuuCAGuCCuCuAAG 130 Scn9a Sa Exon 4_T1 uCGACAuuuuuGGuCCAGuCCG 131 Scn9a Sa Exon 4_T4 CCACCGGACuGGACCAAAAAuG 132 Scn9a Sa Exon 4_T5 GACAAACuGCAuAuuuAuGACC 133 Scn9a Sa Exon 4_T6 AAuAGuGCACAuGAuGAGCAuG 134 Scn9a Sa Exon 4_T7 uGGuCAuAAAuAuGCAGuuuGu 135 Scn9a Sa Exon 5_T1 CuCCuACACAGAAGCCuCuuGC 136 Scn9a Sa Exon 5_T2 uCuuCGuGACCCGuGGAACuGG 137 Scn9a Sa Exon 5_T3 GuuCCACGGGuCACGAAGAAAA 138 Scn9a Sa Exon 5_T4 CuuGCAAGAGGCuuCuGuGuAG 139 Scn9a Sa Exon 5_T5 uuGuGuuuAGGuACACuuuuAC 13 Scn9a Sa Exon 5_T6 ACGACAAAAuCCAGCCAGuuCC 140 Scn9a Sa Exon 5_T7 ACuuuuACuGGAAuAuAuACuu 141 Scn9a Sa Exon 6_T2 uACAAAuuCuGuuAAAuACCuG 142 Scn9a Sa Exon 6_T5 AuuuAAuuCuACAGGuAuuuAA 17 Scn9a Sa Exon 7_T1 CAuGAuCCuGACuGuGuuCuGu 143 Scn9a Sa Exon 7_T2 uuCuAAAGuCuuCuuCACuCuC 144 Scn9a Sa Exon 7_T4 GAGuGAAGAAGACuuuAGAAGu 145 Scn9a Sa Exon 7_T5 AGAAAGCAuAAuGAAuACCCuA 146 Scn9a Sa Exon 7_T6 ACACACuCAGACAGAACACAGu 147 Scn9a Sa Exon 7_T7 uAAuGAAACAuuAGAAAGCAuA 148 Scn9a Sa Exon 7_T8 uCuAAuGuuuCAuuAuuuuCAA 149 Scn9a Sa Exon 8_T1 GAAACCACAAAGGAGAGCAuCu 150 Scn9a Sa Exon 8_T2 CuuuGuGGuuuCAGCACAGAuu 151 Scn9a Sa Exon 8_T3 ACAGAAuAuuuuuAuuACuuGG 152 Scn9a Sa Exon 9_T1 uCAAAGCuCGuGuAGCCAuAAu 18 Scn9a Sa Exon 9_T2 CuCGuGuGuAGuCAGuGuCCAG 14 Scn9a Sa Exon 9_T3 CuGGGAAAACCuuuACCAACAG 153 Scn9a Sa Exon 9_T4 GGuAAAGGuuuuCCCAGuAAuC 154 Scn9a Sa Exon 10_T1 AACAuuGAAGAAGCuAAACAGA 155 Scn9a Sa Exon 10_T2 CAuAuGCCAuGGCAACCACAGC 156 Scn9a Sa Exon 10_T3 GAAGAAGCuAAACAGAAAGAAu 157 Scn9a Sa Exon 11_T1 GCAAuuuGGGuGGuACCuGAuu 158 Scn9a Sa Exon 11_T2 CAGGCAAuuGCAGCGGCAGCGG 11 Scn9a Sa Exon 11_T3 AAGCAGAAuuAuGGGCCuCuCA 159 Scn9a Sa Exon 11_T4 uuuAGCACuuuuAGAGCuCAGu 160 Scn9a Sa Exon 11_T5 GAuGCuGAGAAAuuGuCGAAAu 161 Scn9a Sa Exon 11_T6 AAuAuACAAGuAuuAGGAGAAG 16 Scn9a Sa Exon 12_T1 GAuGuuACuGCuGCGuCGCuCC 12 Scn9a Sa Exon 12_T2 GCCuuGCAGAAAACAAGGAGCC 20 Scn9a Sa Exon 12_T3 AGAAAACAAGGAGCCACGAAuG 162 Scn9a Sa Exon 12_T4 GGAAGAGAuAuAGGAuCuGAGA 163 Scn9a Sa Exon 12_T5 uuCAAAGGCAGAGGAAGAGAuA 164 Scn9a Sa Exon 13_T1 CuAuCuCCuuuCAGAGGAuAuG 165 Scn9a Sa Exon 13_T2 CuCAuuGCuCuCuGuCuGAGGu 15 Scn9a Sa Exon 13_T3 uCCCAACCuCAGACAGAGAGCA 166 Scn9a Sa Exon 13_T4 uuGuAGuuCCuAuCuCCuuuCA 167 Scn9a Sa Exon 14_T1 AuGGAACACCACCCAAuGACuG 168 Scn9a Sa Exon 14_T2 AuAuGGAGAGCAAuuCCAGAuC 169 Scn9a Sa Exon 14_T3 uGAuCuGGAAuuGCuCuCCAuA 170 Scn9a Sa Exon 14_T4 AuGGuAAuuGCAAGAuCuACAA 171 Scn9a Sa Exon 14_T5 uGCuuuuuuCuCCCAGAACuuG 172 Scn9a Sa Exon 14_T6 AuuuCCuAuAGCAAGuACAuuu 173 Scn9a Sa Exon 14_T7 ACAuuuuuGAAuuCCuCAGuCA 174 Scn9a Sa Exon 14_T8 AuuuGCACACAAAuuCuuGAuC 175 Scn9a Sa Exon 14_T9 AAAGuGuAuCuAuuuuAuuGuA 176 Scn9a Sa Exon 14_T10 uACAAuAAAAuAGAuACACuuu 177 Scn9a Sa Exon 15_T1 AAuGGuAuuAAAACuGAuuGCC 178 Scn9a Sa Exon 15_T2 AuAuGAGuAuuuCCAAGuAGGC 19 Scn9a Sa Exon 15_T3 AAACuGAuuGCCAuGGAuCCAu 179 Scn9a Sa Exon 15_T4 ACCuuAGuuuAuGuuuACCAGu 180 Scn9a Sa Exon 15_T5 CAGCCuACuuGGAAAuACuCAu 181 Scn9a Sa Exon 15_T6 GAGCuCuuuCuAGCAGAuGuGG 182 Scn9a Sa Exon 15_T7 uuuuuCuCACuuAGGuCuuuAC

TABLE 3 Names and sequences of SpCas9 guide RNAs targeted to the SCN10A gene (Nav1.8) Nav1.8 SpCas9 gRNAs SEQ ID NO: gRNA Name spacer sequence (5′-3′) 183 Scn10a Sp Exon 1_T2 GACGGAAGuuGuuAGuuuCG 184 Scn10a Sp Exon 1_T3 CAACuuCCGuCGCuuuACuC 185 Scn10a Sp Exon 1_T4 uCGCuuuACuCCGGAGuCAC 186 Scn10a Sp Exon 1_T5 GAACGGAuCuAGAuCCuCCA 187 Scn10a Sp Exon 1_T6 ACGGAAGuuGuuAGuuuCGA 188 Scn10a Sp Exon 1_T7 AACGGAuCuAGAuCCuCCAG 189 Scn10a Sp Exon 1_T8 AGAACGGAuCuAGAuCCuCC 190 Scn10a Sp Exon 1_T9 GuGACuCCGGAGuAAAGCGA 191 Scn10a Sp Exon 1_T10 uuAGuuuCGAGGGAuCCAAu 192 Scn10a Sp Exon 1_T11 GuuAGuuuCGAGGGAuCCAA 193 Scn10a Sp Exon 1_T12 GGCuCCCCGAuCAGuuCuGC 194 Scn10a Sp Exon 1_T14 uAGuuuCGAGGGAuCCAAuG 21 Scn10a Sp Exon 1_T15 GCuCCCCGAuCAGuuCuGCu 195 Scn10a Sp Exon 1_T16 GCuCCCAGCAGAACuGAuCG 28 Scn10a Sp Exon 1_T17 AuCCGuuCuACAGCACACAC 196 Scn10a Sp Exon 1_T18 ACCCGGuGuGuGCuGuAGAA 197 Scn10a Sp Exon 1_T19 AGAACuGAuCGGGGAGCCCC 198 Scn10a Sp Exon 1_T20 uCCGuuCuACAGCACACACC 199 Scn10a Sp Exon 1_T21 CuuuACuCCGGAGuCACuGG 200 Scn10a Sp Exon 1_T22 CACCAuAGAACuuGGGCAGC 201 Scn10a Sp Exon 1_T23 uGGGAGCuCACCAuAGAACu 202 Scn10a Sp Exon 1_T24 ACuGAuCGGGGAGCCCCuGG 203 Scn10a Sp Exon 1_T25 AACCAGCuGCCCAAGuuCuA 204 Scn10a Sp Exon 1_T26 GAACuuGGGCAGCuGGuuGC 205 Scn10a Sp Exon 1_T27 GAAGCAAAuuGCuGCCAAGC 206 Scn10a Sp Exon 1_T28 uCuAuCuCCACCAGuGACuC 207 Scn10a Sp Exon 1_T29 GGGGCCGAGGCuuCuCuuCu 26 Scn10a Sp Exon 1_T30 GAGCuCCCAGCAGAACuGAu 208 Scn10a Sp Exon 2_T1 GGGCCCGAGuGGCACuAAAC 209 Scn10a Sp Exon 2_T2 uuCCCGGuuuAGuGCCACuC 210 Scn10a Sp Exon 2_T3 GGCCCGAGuGGCACuAAACC 211 Scn10a Sp Exon 2_T4 uuuCCCGGuuuAGuGCCACu 212 Scn10a Sp Exon 2_T5 uuAGuGCCACuCGGGCCCuG 213 Scn10a Sp Exon 2_T6 uGAuGGCCGuuCuuCuGAuC 214 Scn10a Sp Exon 2_T7 GAAuAGCCACAGGGCCCGAG 215 Scn10a Sp Exon 2_T8 GuuCuuCuGAuCAGGuuGAA 216 Scn10a Sp Exon 2_T9 AGuGGCACuAAACCGGGAAA 217 Scn10a Sp Exon 2_T10 ACAAAGGGAGGACCAuuuCC 218 Scn10a Sp Exon 4_T1 GGAuuuuAGCGuCAuuACCC 29 Scn10a Sp Exon 4_T2 uCACGuACCuGAGAGAuCCu 219 Scn10a Sp Exon 4_T3 CAuAGAGAuAuACuCACGCC 220 Scn10a Sp Exon 4_T4 GCCAGuuCCAAGGAuCuCuC 221 Scn10a Sp Exon 4_T5 ACCuGAGAGAuCCuuGGAAC 222 Scn10a Sp Exon 5_T1 GCACAGCAAuAGAuCuCCGu 223 Scn10a Sp Exon 5_T2 uAAGAACuCuGAAuGuCCGC 224 Scn10a Sp Exon 5_T3 AuAGAuCuCCGuGGGAuCuC 225 Scn10a Sp Exon 5_T4 GGCACAGCAAuAGAuCuCCG 226 Scn10a Sp Exon 6_T2 GGGCCCCCACAAuGACCuuC 227 Scn10a Sp Exon 6_T3 CGCAGGCCuGAAGGuCAuuG 228 Scn10a Sp Exon 6_T4 GuGGGGCuGCAACuCuuCAA 229 Scn10a Sp Exon 6_T7 GCAGGCCuGAAGGuCAuuGu 230 Scn10a Sp Exon 6_T8 GGuGGGGCuGCAACuCuuCA 231 Scn10a Sp Exon 6_T9 uCAuCuuCCCGCAGGCCuGA 232 Scn10a Sp Exon 6_T10 GAAGAGuuGCAGCCCCACCA 233 Scn10a Sp Exon 7_T1 GACCCCuuACuGuGuGGCAA 234 Scn10a Sp Exon 7_T2 AGAuCCAuuGCCACACAGuA 235 Scn10a Sp Exon 7_T3 uGuGGCAAuGGAuCuGACuC 236 Scn10a Sp Exon 7_T4 GuGGCAAuGGAuCuGACuCA 237 Scn10a Sp Exon 7_T5 GAuCCAuuGCCACACAGuAA 25 Scn10a Sp Exon 7_T6 ACuuCuGACCCCuuACuGuG 238 Scn10a Sp Exon 7_T7 AuCCAuuGCCACACAGuAAG 239 Scn10a Sp Exon 8_T1 ACuGuuCCGCCuCAuGACAC 240 Scn10a Sp Exon 8_T2 CuGGGAACGCCuCuACCAGC 241 Scn10a Sp Exon 8_T3 CCGGGuuGuCAGAAGuuuuA 242 Scn10a Sp Exon 8_T4 GCCuCAuGACACAGGAuuCC 243 Scn10a Sp Exon 8_T5 AGCuCAGuACCuGCuGGuAG 244 Scn10a Sp Exon 8_T6 uuAAGGCAGAuAuAACCAuC 245 Scn10a Sp Exon 8_T7 AAGCuGGuGuAGuuAAAAuC 246 Scn10a Sp Exon 8_T8 uCAuGAGGCGGAACAGuGAG 247 Scn10a Sp Exon 8_T9 CCuuAAAACuuCuGACAACC 248 Scn10a Sp Exon 8_T10 GAAuGCAGCuCAGuACCuGC 249 Scn10a Sp Exon 9_T1 GGCCCuCGAGAuGCuCCGGA 22 Scn10a Sp Exon 9_T2 uGuAGuCACCAuGGCGuAuG 250 Scn10a Sp Exon 9_T3 GuuCuGCuCCuCAuACGCCA 251 Scn10a Sp Exon 9_T4 CuCCuuCCGGAGCAuCuCGA 252 Scn10a Sp Exon 9_T5 CuACCuGGuCAACuuGAuCu 253 Scn10a Sp Exon 9_T6 GCuCCuuCCGGAGCAuCuCG 254 Scn10a Sp Exon 9_T7 CGAGAuGCuCCGGAAGGAGC 255 Scn10a Sp Exon 9_T8 AGGAGGCCCuCGAGAuGCuC 256 Scn10a Sp Exon 9_T9 uuuGuGCuCGuAAuCuuCCu 257 Scn10a Sp Exon 9_T10 GGAGCAuCuCGAGGGCCuCC 258 Scn10a Sp Exon 9_T11 GGCGuAuGAGGAGCAGAACC 259 Scn10a Sp Exon 9_T12 uuuuGuGCuCGuAAuCuuCC 260 Scn10a Sp Exon 9_T13 uGACCAGGuAGAAAGAuCCC 261 Scn10a Sp Exon 9_T14 GAuCuuGGCuGuAGuCACCA 262 Scn10a Sp Exon 10_T1 ACCAuCCuGCGCuGGuuGuA 263 Scn10a Sp Exon 10_T2 CuGAuCCuuACAACCAGCGC 30 Scn10a Sp Exon 10_T3 CGCAGGuGCuAGCAGCACuA 264 Scn10a Sp Exon 10_T4 uCGCAGGuGCuAGCAGCACu 265 Scn10a Sp Exon 10_T5 GGuuAAAGGuGAuCCAuuGu 266 Scn10a Sp Exon 10_T6 AGCCuCuuACCAuCCuGCGC 24 Scn10a Sp Exon 10_T7 uCCuuACAACCAGCGCAGGA 267 Scn10a Sp Exon 10_T9 AGGuuAAAGGuGAuCCAuuG 268 Scn10a Sp Exon 10_T10 uGGuuGuAAGGAuCAGAGCG 269 Scn10a Sp Exon 10_T13 GuGGAGCCCuCuGACACuCu 270 Scn10a Sp Exon 11_T1 GCuCACuAGuGGGCGGCGGu 271 Scn10a Sp Exon 11_T2 GGAAAACGCCGGGCuAGuCA 272 Scn10a Sp Exon 11_T3 ACuAGCCCGGCGuuuuCCAG 273 Scn10a Sp Exon 11_T4 ACCGAGACAuCGACAGCuCC 274 Scn10a Sp Exon 11_T5 CCCGGCGuuuuCCAGAGGCG 275 Scn10a Sp Exon 11_T6 GAGAGCCCCGAuGGCuuuCG 276 Scn10a Sp Exon 11_T7 CGAuGGCuuuCGuGGuCuCC 497 Scn10a Sp Exon 11_T8 GCAAGCuCACuAGuGGGCGG 277 Scn10a Sp Exon 11_T9 CCuCGCCuCuGGAAAACGCC 27 Scn10a Sp Exon 11_T10 CCGAGACAuCGACAGCuCCA 278 Scn10a Sp Exon 11_T11 CGAGACAuCGACAGCuCCAG 279 Scn10a Sp Exon 11_T12 GCCuCGCCuCuGGAAAACGC 280 Scn10a Sp Exon 11_T13 GGGAGuGAGAuAuCuCGGCC 281 Scn10a Sp Exon 11_T14 GGAGACCACGAAAGCCAuCG 282 Scn10a Sp Exon 11_T15 CGAGAuAuCuCACuCCCuGA 283 Scn10a Sp Exon 11_T16 CCCACuAGuGAGCuuGCCCC 284 Scn10a Sp Exon 11_T17 CCAGGGGCAAGCuCACuAGu 285 Scn10a Sp Exon 11_T18 GAGuGAGAuAuCuCGGCCAG 286 Scn10a Sp Exon 11_T19 CCGAGAuAuCuCACuCCCuG 287 Scn10a Sp Exon 11_T20 CuCGGCCAGGGGACCGGAAA 288 Scn10a Sp Exon 11_T21 AGAuAuCuCGGCCAGGGGAC 289 Scn10a Sp Exon 11_T22 GuGuuCCAuuuCCGGuCCCC 290 Scn10a Sp Exon 11_T23 uCCAGGGGCAAGCuCACuAG 291 Scn10a Sp Exon 11_T24 GGGGCAAGCuCACuAGuGGG 292 Scn10a Sp Exon 11_T25 GGAGuGAGAuAuCuCGGCCA 293 Scn10a Sp Exon 11_T26 uGGAGACCACGAAAGCCAuC 294 Scn10a Sp Exon 11_T27 GGCGAGGCCuAGAAAAGACu 295 Scn10a Sp Exon 11_T28 CCCuGGAGCuGuCGAuGuCu 296 Scn10a Sp Exon 11_T29 CuGGAGACCACGAAAGCCAu 297 Scn10a Sp Exon 11_T30 uCuuuuCuAGGCCuCGCCuC 298 Scn10a Sp Exon 11_T31 AGGCGAGGCCuAGAAAAGAC 299 Scn10a Sp Exon 11_T32 CCuCAGGGAGuGAGAuAuCu 300 Scn10a Sp Exon 11_T33 GuuGAGGAAGAGGGCuuCuA 301 Scn10a Sp Exon 11_T34 uuCuAGGGAGGGGGCCuuGC 302 Scn10a Sp Exon 12_T1 uCuCAACAGGCAuuCGAuGC 303 Scn10a Sp Exon 12_T2 AuGAACCuuuCCGGGCCCAA 304 Scn10a Sp Exon 12_T3 GCACuuACCCuCAAGGACGG 305 Scn10a Sp Exon 12_T4 uAuCAuAACCuCCGuCCuuG 306 Scn10a Sp Exon 12_T5 uGAACCuuuCCGGGCCCAAA 307 Scn10a Sp Exon 12_T6 AuCAuAACCuCCGuCCuuGA 308 Scn10a Sp Exon 12_T7 AuuGCCCuuuGGGCCCGGAA 309 Scn10a Sp Exon 12_T8 GuGAGCAGCACuuACCCuCA 310 Scn10a Sp Exon 12_T9 GCAGCACuuACCCuCAAGGA 311 Scn10a Sp Exon 12_T10 CACuCAuuGCCCuuuGGGCC 312 Scn10a Sp Exon 12_T12 GACAACACuCAuuGCCCuuu 313 Scn10a Sp Exon 12_T13 AuACuuAGAuGAACCuuuCC 314 Scn10a Sp Exon 12_T14 uGACAACACuCAuuGCCCuu 315 Scn10a Sp Exon 13_T1 CACuCACGAuGuuGCCuAuC 316 Scn10a Sp Exon 13_T4 uuCGAAGCCAuGCuCCAGAu 317 Scn10a Sp Exon 13_T5 CACCAuCACCuuGuGCAuCG 318 Scn10a Sp Exon 13_T6 ACACuAuAAuGCAGAACuCG 319 Scn10a Sp Exon 13_T8 CACCACGAuGCACAAGGuGA 320 Scn10a Sp Exon 13_T9 CGuGGuGAACACCAuCuuCA 321 Scn10a Sp Exon 13_T10 GGAGCAuGGCuuCGAAGGuA 322 Scn10a Sp Exon 13_T11 uAuCuGGAGCAuGGCuuCGA 323 Scn10a Sp Exon 13_T12 uGGAGCAuGGCuuCGAAGGu 324 Scn10a Sp Exon 13_T13 CGAAGGuAGGGCuCAuGCCA 325 Scn10a Sp Exon 13_T14 GGuGuuCACCACGAuGCACA 326 Scn10a Sp Exon 13_T15 ACAAGCuGGuCAAGCAGGGu 327 Scn10a Sp Exon 13_T16 GAuGuuGCCuAuCuGGAGCA 328 Scn10a Sp Exon 13_T17 uuCAuGGCCAuGGAGCACCA 329 Scn10a Sp Exon 13_T18 uCuuGAGCuuCACCCACAuG 330 Scn10a Sp Exon 13_T19 CuGGGAuuGCuGCCCCAuGu 331 Scn10a Sp Exon 13_T20 uGuCuuGAGCuuCACCCACA 332 Scn10a Sp Exon 13_T21 GuGAuGGuGAGCuCuGCAAA 333 Scn10a Sp Exon 13_T22 GGuGAuGGuGAGCuCuGCAA 334 Scn10a Sp Exon 14_T1 uGuGCuGCGGAGCuuCCGCu 335 Scn10a Sp Exon 14_T2 GAAAuAAuAGuAuGGGuCGA 23 Scn10a Sp Exon 14_T3 GGAAGCuCCGCAGCACAGAC 336 Scn10a Sp Exon 14_T4 ACuGuGAGuCuGCuAGAGCu 337 Scn10a Sp Exon 14_T5 CACuGuGAGuCuGCuAGAGC

TABLE 4  Names and sequences of SaCas9 guide RNAs targeted to the SCN10A gene (Nav1.8) Nav1.8 SaCas9 gRNAs SEQ ID NO: gRNA Name spacer sequence (5′-3′) 338 Scn10a Sa Exon 1_T1 GCGACGGAAGuuGuuAGuuuCG 38 Scn10a Sa Exon 1_T3 AACAACuuCCGuCGCuuuACuC 339 Scn10a Sa Exon 1_T4 GuuAGuuuCGAGGGAuCCAAuG 340 Scn10a Sa Exon 1_T5 CuuACCCGGuGuGuGCuGuAGA 341 Scn10a Sa Exon 1_T6 AGAACuGAuCGGGGAGCCCCuG 32 Scn10a Sa Exon 1_T7 uAGAuCCGuuCuACAGCACACA 342 Scn10a Sa Exon 1_T8 uCuCuAuCuCCACCAGuGACuC 343 Scn10a Sa Exon 1_T9 AAuGAGAAGAuGGAAuuCCCCA 344 Scn10a Sa Exon 2_T1 AAGGACuGAAuAGCCACAGGGC 345 Scn10a Sa Exon 2_T2 uCuuCuGAuCAGGuuGAAAGGA 346 Scn10a Sa Exon 3_T1 CuGACCuuCCAGAGAAAAuuGA 347 Scn10a Sa Exon 3_T2 CAAuuuuCuCuGGAAGGuCAGu 348 Scn10a Sa Exon 3_T3 CGAACuGACCuuCCAGAGAAAA 349 Scn10a Sa Exon 4_T2 CuGGCAAGAGGAuuuuGuCuAA 350 Scn10a Sa Exon 4_T3 ACGCuAAAAuCCAGCCAGuuCC 351 Scn10a Sa Exon 4_T4 CCuGAGAGAuCCuuGGAACuGG 37 Scn10a Sa Exon 4_T5 AuuuuAGCGuCAuuACCCuGGC 352 Scn10a Sa Exon 4_T7 GCCuuGAuAAAGAuACuGGCAA 353 Scn10a Sa Exon 5_T1 uuGGCACAGCAAuAGAuCuCCG 354 Scn10a Sa Exon 5_T2 GGAuCuCAGGCCuGCGGACAuu 355 Scn10a Sa Exon 5_T4 uGuuuuuAAuGCuCuAAGAACu 356 Scn10a Sa Exon 6_T1 CuCCACuCACGuuuuCuGuGAG 357 Scn10a Sa Exon 6_T3 AAACACuuAGGCAGAAGAuGGu 358 Scn10a Sa Exon 6_T4 CAuCAGCCAGuuuCuuCACuGA 359 Scn10a Sa Exon 6_T5 AACuACuCAuCuCACAGAAAAC 360 Scn10a Sa Exon 6_T6 GuCACAuCAGCCAGuuuCuuCA 361 Scn10a Sa Exon 6_T7 CAACCuCAAAAAuAAAuGuGuC 362 Scn10a Sa Exon 6_T8 uuuAuuuuuGAGGuuGCCCuuG 363 Scn10a Sa Exon 7_T2 uCAGAuCCAuuGCCACACAGuA 364 Scn10a Sa Exon 7_T3 CuGuGuGGCAAuGGAuCuGACu 365 Scn10a Sa Exon 7_T4 GuGGCAAuGGAuCuGACuCAGG 366 Scn10a Sa Exon 7_T5 uCuGACCCCuuACuGuGuGGCA 367 Scn10a Sa Exon 8_T1 ACCuGCuGGuAGAGGCGuuCCC 368 Scn10a Sa Exon 8_T2 CuCACuGuuCCGCCuCAuGACA 369 Scn10a Sa Exon 8_T3 CuGCCuuAAAACuuCuGACAAC 370 Scn10a Sa Exon 8_T4 uCAAAGCuGGuGuAGuuAAAAu 33 Scn10a Sa Exon 8_T5 AGuGAGAGGAAAGCCCAAGCAA 371 Scn10a Sa Exon 9_T1 uuuuuuGuGCuCGuAAuCuuCC 372 Scn10a Sa Exon 9_T3 uAuAGAuuuuCCCAGAAGuCCu 373 Scn10a Sa Exon 10_T1 GCuCuGAuCCuuACAACCAGCG 374 Scn10a Sa Exon 10_T2 CuuCGCAGGuGCuAGCAGCACu 375 Scn10a Sa Exon 10_T3 CuuACCAuCCuGCGCuGGuuGu 376 Scn10a Sa Exon 10_T4 GCGCuGGuuGuAAGGAuCAGAG 377 Scn10a Sa Exon 10_T5 ACAACCuCuCuCCACuCCCACA 378 Scn10a Sa Exon 10_T6 GAGGuuAAAGGuGAuCCAuuGu 379 Scn10a Sa Exon 10_T7 AGAGAAGGCAuAGAAuAAAGCC 380 Scn10a Sa Exon 10_T8 AAAAuGCCAGuGAGAGAAGGCA 31 Scn10a Sa Exon 11_T1 CCCuGGAGCuGuCGAuGuCuCG 39 Scn10a Sa Exon 11_T2 GCCGAGAuAuCuCACuCCCuGA 381 Scn10a Sa Exon 11_T3 AAGCCAuCGGGGCuCuCuGCuG 382 Scn10a Sa Exon 11_T4 uCCAuCAuCuGuGACuCCCuCA 40 Scn10a Sa Exon 11_T5 uGGuGuuCAuCuuCuCCAuGCC 383 Scn10a Sa Exon 11_T7 uCGGGGCuCuCuGCuGCuGGGu 384 Scn10a Sa Exon 11_T8 uCCAuGCCuGGAGuCAGGGuuG 385 Scn10a Sa Exon 11_T9 uCCCuGAGGGAGuCACAGAuGA 386 Scn10a Sa Exon 11_T10 uCAuCuuCuCCAuGCCuGGAGu 387 Scn10a Sa Exon 12_T1 CAGuAuCAuAACCuCCGuCCuu 34 Scn10a Sa Exon 12_T2 ACCuuuCCGGGCCCAAAGGGCA 388 Scn10a Sa Exon 12_T4 GAAAGuCuuCuuuuGuCCuGCA 389 Scn10a Sa Exon 12_T5 CAAAAGAAGACuuuCuuGuCAG 390 Scn10a Sa Exon 13_T1 CCACACuAuAAuGCAGAACuCG 391 Scn10a Sa Exon 13_T2 CAuGCuCCAGAuAGGCAACAuC 392 Scn10a Sa Exon 13_T3 AGGGAuCCGuCACAAGCCCAAA 393 Scn10a Sa Exon 13_T4 uGAuCuGGGAuuGCuGCCCCAu 394 Scn10a Sa Exon 13_T5 CuuGuCuCAGAAGuAuCuGAuC 395 Scn10a Sa Exon 13_T6 GACAAuuCuCuuuGGGCuuGuG 396 Scn10a Sa Exon 13_T7 CuuCuGAGACAAGCuGGuCAAG 397 Scn10a Sa Exon 13_T8 AAGGuGAuGGuGAGCuCuGCAA 398 Scn10a Sa Exon 13_T9 uGGGCACuuCuGuuCAGACuCC 35 Scn10a Sa Exon 14_T1 CuuuGACuGCAuCAuCGuCACu 36 Scn10a Sa Exon 14_T2 CACuuCuuCuGGAAAuAAuAGu 399 Scn10a Sa Exon 14_T3 GuAAAAAAuAuGGuAAAGACCu 400 Scn10a Sa Exon 14_T4 AuACuAuuAuuuCCAGAAGAAG

TABLE 5  Names, sequences and targeted exons of primers used for sequencing analysis of CRISPR-mediated editing of the SCN9A gene (Nav1.7) Nav1.7 NGS Primers SEQ ID NO: Primer Sequence Exon 401 Nav1-7-Exon-2-1-NGS-F1 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCA 2-1 TCCAGGCCTCTTATGTGAGGAG 402 Nav1-7-Exon-2-1-NGS-R1 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGA TAGATGAAGGGCAGCTGTTTGC 403 Nav1-7-Exon-2-2-NGS-F1 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTG 2-2 AACAACGCATTGCTGAAAGA 404 Nav1-7-Exon-2-2-NGS-R1 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGT TTTATACAGAAGGAAGCCAACAG 405 Nav1-7-Exon-3-NGS-F2 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGA 3 ACTGCTGATATTGATGTGAAAAA 406 Nav1-7-Exon-3-NGS-R2 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGA AAATAGCAAAAATTACACCATAAAGT 407 Nav1-7-Exon-4-NGS-F2 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTC 4 CTCAAATATTTCAAATTCCCACTGT 408 Nav1-7-Exon-4-NGS-R2 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGT CCCCATCTTCATAAATGCAGTAAC 409 Nav1-7-Exon-5-NGS-F1 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGA 5 AAGATTTACATGGTGGTTGTATTCTT 410 Nav1-7-Exon-5-NGS-R1 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGC TGTGCTGCCTGAGATTTTCAT 411 Nav1-7-Exon-6-NGS-F2 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGA 6 GCCCCAAACGTAGAAAATACCT 412 Nav1-7-Exon-6-NGS-R2 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGC TCCCAAATAGTTGGAGTTATGAGT 413 Nav1-7-Exon-7-1-NGS-F2 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGT 7-1 TTCGATTCAGAGGCTTTATGTC 414 Nav1-7-Exon-7-1-NGS-R2 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGC TCTCTAGGGTATTCATTATGCTTTCT 415 Nav1-7-Exon-7-2-NGS-F2 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGG 7-2 AAGCTTTCTGATGTCATGATCC 416 Nav1-7-Exon-7-2-NGS-R2 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGG CAACATTTCATTATTAAAAGAGAGCA 417 Nav1-7-Exon-8-NGS-F1 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGG 8 GACCAGGCCTGAATTTGTAG 418 Nav1-7-Exon-8-NGS-R1 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGT TTGCAAACTGACTGAACATTCT 419 Nav1-7-Exon-9-NGS-F3 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGT 9 CCTGAAGACACTCTCACCT 420 Nav1-7-Exon-9-NGS-R3 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGC AGGCTCTTAACATACACCAGG 421 Nav1-7-Exon-10-1-NGS-F1 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTG 10-1 CTCATGCCTGTCAAATTGAAATA 422 Nav1-7-Exon-10-1-NGS-R1 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGA CATCTGTTGAAATTCTAATTCTTTCTGT 423 Nav1-7-Exon-10-2-NGS-F4 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCT 10-2 AGACGCTGCGTGCTGCT 424 Nav1-7-Exon-10-2-NGS-R4 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGA AGGCCAAGCATATACCGCAGA 425 Nav1-7-Exon-11-1-NGS-F3 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAT 11-1 GTCCTGTCCTAGGGTTTCCT 426 Nav1-7-Exon-11-1-NGS-R3 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGT CAGCATCTCCCTTTTCCTCTC 427 Nav1-7-Exon-11-2-NGS-F1 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGG 11-2 GCAGCGGCTGAATATACAAGT 428 Nav1-7-Exon-11-2-NGS-R1 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGC TCGCCTATGCCCTTCGAC 429 Nav1-7-Exonl 1-3-NGS-F3 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGA 11-3 GAATCAAAAGAAGCTCTCCAGTG 430 Nav1-7-Exonl 1-3-NGS-R3 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGT CACTCACTATCCTCTCCCGA 431 Nav1-7-Exon12-1-NGS-F6 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGA 12-1 GTGTACTTCTATCAGTAGGTGCTT 432 Nav1-7-Exon12-1-NGS-R6 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGG ACCTACTGGCTTGGCTGAT 433 Nav1-7-Exon-12-2-NGS-F2 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGA 12-2 AGCAGCAGAACAAGTCTTTTTAGTT 434 Nav1-7-Exon-12-2-NGS-R2 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGA CCAGGGAGACCACACCGT 435 Nav1-7-Exon12-3-NGS-F6 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAC 12-3 AGCATTTTTGGAGACAATGAGA 436 Nav1-7-Exon12-3-NGS-R6 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGA TGCCTGAGCTATGTAAAACGTC 437 Nav1-7-Exon-13-1-NGS-F1 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCC 13-1 CAGCAATCTAGGCTCTACT 438 Nav1-7-Exon-13-1-NGS-R1 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGC CTTCCACAGTGTTTGTTAATATGC 439 Nav1-7-Exon-13-2-NGS-F4 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTC 13-2 AAATACACAAGAAAAGGCGTTG 440 Nav1-7-Exon-13-2-NGS-R4 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGA CAATTCCATCAGTATCCATTGGT 441 Nav1-7-Exon-14-1-NGS-F1 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGG 14-1 GTTAGGAGTGAAACAGACAAATGG 442 Nav1-7-Exon-14-1-NGS-R1 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGT GGTGTTCCATAGCCATAAATAATGTG 443 Nav1-7-Exon-14-2-NGS-F2 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTC 14-2 TTGATCTGGAATTGCTCTCCAT 444 Nav1-7-Exon-14-2-NGS-R2 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGA CAATGATGACAACTAAAAAGAGAAACT 445 Nav1-7-Exon-15-1-NGS-F2 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAT 15-1 CATTGTGTTGATTTCCTGTTTTCT 446 Nav1-7-Exon-15-1-NGS-R2 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGC CAGTCTGAATGATCGCAGAAC 447 Nav1-7-Exon-15-2-NGS-F2 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTG 15-2 CAGCTGAAATGGTATTAAAACTGA 448 Nav1-7-Exon-15-2-NGS-R2 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGT GCAAAACCAAAGAAATACCCCTT

TABLE 6  Names, sequences and targeted exons of primers used for sequencing analysis of CRISPR-mediated editing of the SCN10A gene (Nav1.8) Nav1.8 NGS Primers SEQ ID NO: Primer Sequence Exon 449 Nav1-8-Exon-1-1-NGS-F4 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGC  1-1 TGTCACCTCTCTGTGGTT 450 Nav1-8-Exon-1-1-NGS-R4 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGT AGAACTTGGGCAGCTGGTT 451 Nav1-8-Exon-1-2-NGS-F1 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCC  1-2 AAGCAGGGAACAAAGAAA 452 Nav1-8-Exon-1-2-NGS-R1 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGC CGAGACTTCCTCTCCAAGA 453 Nav1-8-Exon-2-1-NGS-F3 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGC 2 CTGAGATAATGCCTCTCATGT 454 Nav1-8-Exon-2-1-NGS-R3 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGA CTGGACACAGTAGGCAAGG 455 Nav1-8-Exon-3-1-NGS-F1 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTG 3 TAATCATTCAGCATCAAGGTG 456 Nav1-8-Exon-3-1-NGS-R1 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGA CAAAGACTGCCAAGTGAAGGA 457 Nav1-8-Exon-4-1-NGS-F3 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCA 4 TCCCTCCCTCCTGGAAGA 458 Nav1-8-Exon-4-1-NGS-R3 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGC CCCTCTCCTATCACACATGC 459 Nav1-8-Exon-5-1-NGS-F3 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGA 5 GGCTAATGATACCCCAGGT 460 Nav1-8-Exon-5-1-NGS-R3 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGA GTCTTTGCCCTGGAACCTT 461 Nav1-8-Exon-6-1-NGS-F4 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGT  6-1 GTGCTCCGTGTGTGCTAC 462 Nav1-8-Exon-6-1-NGS-R4 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGA ACCAGACCTTGGTCCCTATG 463 Nav1-8-Exon-6-2-NGS-F4 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCT  6-2 TCAAGGGCAACCTCAAAA 464 Nav1-8-Exon-6-2-NGS-R4 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGA TTTCCTTGCAAGAGGGATG 465 Nav1-8-Exon-7-1-NGS-F1 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAT 7 TGCATTCACCACACAAGG 466 Nav1-8-Exon-7-1-NGS-R1 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGA TGCCAAGGACAAGATGGAG 467 Nav1-8-Exon-8-1-NGS-F1 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTG 8 GGCTACCTTGTCTGCAAT 468 Nav1-8-Exon-8-1-NGS-R1 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGA GCCTCCAACCAAGTCTGC 469 Nav1-8-Exon-9-1-NGS-F2 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCC  9-1 TGGAGGAGGCTGACTTAAA 470 Nav1-8-Exon-9-1-NGS-R2 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGA GGGCCTCCTGGAACTTCTT 471 Nav1-8-Exon-9-2-NGS-F4 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTG  9-2 CAGACCCTGAGGACTTCT 472 Nav1-8-Exon-9-2-NGS-R4 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGT GGACAGTCTGCAACCTTCT 473 Nav1-8-Exon-10-1-NGS-F5 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTC 10  ATGCTAAGTCCAAGCAAATACT 474 Nav1-8-Exon-10-1-NGS-R5 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGG CAGCTGCAATGGTGGGTAA 475 Nav1-8-Exon-11-1-NGS-F6 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTT 11-1 CCAGCCTTCTTGCTCCTTT 476 Nav1-8-Exon-11-1-NGS-R6 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGA GTCAGGGTTGCTGGGTTGA 477 Nav1-8-Exon-11-2-NGS-F3 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCC 11-2 CTGAGGGAGTCACAGATG 478 Nav1-8-Exon-11-2-NGS-R3 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGG CTCAAGGCTTCTAGGTGGA 479 Nav1-8-Exon-12-1-NGS-F2 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTT 12 TGCCATGAAGATGTCAGG 480 Nav1-8-Exon-12-1-NGS-R2 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGT GCTCACATGGGAATTCATC 481 Nav1-8-Exon-13-1-NGS-F2 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGA 13-1 GAGGATGACCGCAGAATTG 482 Nav1-8-Exon-13-1-NGS-R2 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGA TGCACAAGGTGATGGTGAG 483 Nav1-8-Exon-13-2-NGS-F4 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCT 13-2 TGACCAGCTTGTCTCAGAAG 484 Nav1-8-Exon-13-2-NGS-R4 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGA CTGCACCCTGCCATCAT 485 Nav1-8-Exon-14-1-NGS-F1 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAC 14-1 CCCACAGATCCCACTGT 486 Nav1-8-Exon-14-1-NGS-R1 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGC ACAGACAGGCTTCCCTTCTT 487 Nav1-8-Exon-14-2-NGS-F1 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTG 14-2 CTGAAATGGTCTTCAAAATC 488 Nav1-8-Exon-14-2-NGS-R1 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGT GAATCTGGGTGGGAGTTTC

TABLE 7 Total list of 1,471 putative off-target sites predicted across the 40 guides chr start end chr start end chr start end chr1 101673862 101673883 chr6 81389456 81389478 chr8 18547775 18547797 chr1 181263539 181263560 chr7 116885434 116885456 chr9 121398998 121399019 chr1 230060685 230060706 chr8 101163760 101163782 chr9 90536880 90536903 chr1 57212888 57212911 chr8 102040037 102040058 chrX 154575630 154575653 chr10 129962874 129962895 chr8 32017421 32017443 chrX 154628777 154628800 chr10 84935685 84935706 chr9 134428917 134428939 chr1 218298983 218299004 chr10 8530131 8530152 chr9 41739585 41739607 chr1 42210368 42210390 chr11 11657568 11657590 chr9 61575358 61575380 chr1 58885286 58885307 chr11 118434780 118434801 chr9 67636882 67636904 chr1 99737182 99737204 chr11 19769579 19769600 chrX 103932345 103932367 chr10 119436660 119436681 chr11 44591707 44591728 chrX 149687593 149687615 chr10 78303428 78303451 chr11 60420702 60420725 chrX 77969229 77969250 chr11 105632443 105632465 chr11 79356353 79356374 chrY 17093954 17093976 chr11 49938853 49938876 chr11 81605512 81605533 chr1 106130660 106130681 chr12 103863493 103863514 chr12 118776061 118776082 chr1 17346264 17346285 chr12 92649761 92649783 chr12 54000745 54000768 chr1 191298075 191298097 chr13 112185105 112185127 chr12 95630598 95630619 chr1 236890187 236890208 chr13 29354998 29355019 chr13 110180589 110180611 chr1 244884251 244884272 chr14 56541130 56541151 chr13 40148712 40148733 chr10 79776620 79776641 chr14 63594134 63594155 chr13 60239631 60239653 chr10 87298770 87298791 chr15 79057189 79057210 chr13 66558377 66558400 chr10 87439176 87439197 chr16 1024374 1024397 chr14 104081758 104081780 chr11 133566119 133566141 chr17 57649035 57649057 chr14 104550954 104550976 chr11 18269330 18269351 chr2 142368179 142368200 chr14 70672101 70672122 chr11 31057715 31057736 chr2 166286371 166286393 chr14 72884578 72884599 chr11 86679889 86679910 chr20 24803468 24803489 chr14 76371571 76371593 chr13 26095101 26095122 chr20 24803468 24803490 chr15 75765848 75765871 chr13 88051002 88051025 chr20 25276655 25276676 chr16 51266796 51266817 chr15 54619157 54619179 chr20 57304528 57304551 chr16 66970656 66970677 chr16 78175674 78175695 chr20 63700780 63700801 chr17 38736344 38736365 chr16 85698295 85698317 chr3 12181396 12181417 chr17 5662660 5662682 chr18 4435202 4435223 chr3 127363721 127363742 chr17 77914635 77914656 chr18 63953514 63953535 chr3 73435663 73435686 chr19 14021634 14021656 chr18 75626393 75626414 chr4 148797789 148797810 chr2 127092215 127092238 chr19 16143760 16143782 chr4 98007120 98007141 chr2 134821956 134821977 chr19 3636896 3636918 chr5 141787884 141787905 chr2 178940831 178940853 chr2 102073639 102073660 chr5 155635156 155635177 chr20 20432289 20432311 chr2 111189580 111189601 chr6 104164934 104164956 chr20 58601338 58601359 chr2 15761798 15761820 chr7 139583922 139583943 chr21 35410460 35410482 chr2 178287459 178287480 chr7 142193746 142193769 chr22 39957473 39957496 chr2 81854796 81854819 chr7 47045289 47045310 chr3 119381654 119381676 chr2 96711649 96711670 chr7 99403691 99403712 chr3 140431388 140431409 chr20 46241748 46241769 chr9 89919212 89919233 chr3 38755787 38755809 chr20 58037844 58037865 chr9 97393314 97393335 chr4 109874522 109874543 chr20 61210620 61210641 chrX 101837971 101837993 chr4 135986289 135986310 chr21 31457963 31457984 chrX 102321859 102321881 chr4 162888411 162888432 chr3 38771311 38771333 chrX 102365234 102365256 chr4 95257196 95257217 chr3 67894307 67894329 chrX 42045623 42045644 chr5 91935098 91935119 chr5 150570292 150570313 chrX 95359671 95359693 chr6 106430770 106430791 chr5 163518469 163518491 chr1 106905030 106905051 chr6 154267869 154267892 chr6 41702022 41702043 chr1 187369871 187369893 chr7 14664086 14664107 chr6 42338824 42338847 chr1 217283942 217283964 chr7 41078138 41078159 chr8 133739469 133739491 chr1 67514969 67514990 chr7 56759248 56759270 chr8 141160774 141160795 chr1 73958112 73958133 chr7 63816239 63816261 chr8 20158237 20158258 chr10 131976154 131976175 chr8 117647612 117647633 chr8 23430667 23430688 chr10 4150457 4150479 chr8 19663295 19663316 chr9 130371904 130371925 chr10 45658400 45658421 chr8 26849329 26849350 chr9 136746114 136746135 chr10 54308353 54308374 chr8 46389287 46389309 chr9 31741140 31741162 chr10 86859468 86859489 chr8 500777 500799 chrX 38085667 38085689 chr10 95305836 95305858 chr8 67346602 67346623 chrX 88090240 88090261 chr12 16683841 16683862 chr9 109783158 109783179 chr1 104446115 104446136 chr12 17604379 17604400 chr9 130987364 130987385 chr1 182332402 182332423 chr12 83263173 83263195 chr9 132448490 132448511 chr1 183710217 183710238 chr16 3841926 3841947 chr9 3023108 3023129 chr1 208748851 208748872 chr18 71712771 71712794 chr9 70925755 70925777 chr1 240969802 240969823 chr2 155442093 155442116 chr9 78073257 78073279 chr1 26574711 26574733 chr2 166286585 166286607 chrX 130376370 130376393 chr1 30456582 30456603 chr2 171528653 171528674 chrX 39650052 39650074 chr1 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144505858 144505879 chr1 44003243 44003264 chr5 38374328 38374349 chr7 28106012 28106033 chr1 60484489 60484511 chr5 51582987 51583008 chr7 5261424 5261446 chr1 77272589 77272611 chr5 51584136 51584157 chr7 56972149 56972171 chr1 99805299 99805321 chr5 5684323 5684344 chr7 63604777 63604799 chr10 7234091 7234112 chr5 95184977 95184998 chr9 11380189 11380210 chr11 124639215 124639237 chr6 10345815 10345837 chrX 74835062 74835083 chr11 128565919 128565940 chr6 111939969 111939990 chr1 113789694 113789716 chr11 32183988 32184009 chr6 144851102 144851123 chr1 151492228 151492250 chr11 98824964 98824985 chr6 169660524 169660545 chr1 17434745 17434767 chr12 52539784 52539807 chr6 44791613 44791636 chr1 237232784 237232806 chr13 53239100 53239123 chr6 54214841 54214862 chr1 51343936 51343958 chr13 67710449 67710471 chr6 85108470 85108493 chr1 62060283 62060305 chr14 101542861 101542882 chr7 101293356 101293377 chr10 12202452 12202474 chr14 94101963 94101986 chr7 101769888 101769909 chr10 34500129 34500150 chr15 53389064 53389085 chr7 71436053 71436074 chr10 908595 908617 chr15 59609984 59610006 chr7 77258325 77258348 chr10 97430628 97430649 chr16 83718885 83718908 chr8 103188339 103188360 chr11 131800586 131800608 chr17 13619613 13619634 chr8 58208948 58208971 chr11 45059654 45059675 chr18 1302211 1302234 chr8 90410702 90410725 chr11 46293596 46293618 chr18 34382228 34382250 chr9 16091078 16091101 chr11 67532922 67532943 chr2 136683476 136683499 chr9 87763613 87763634 chr11 68473701 68473723 chr2 166280389 166280411 chr9 89771805 89771827 chr11 76869199 76869221 chr20 21609437 21609459 chrX 114583997 114584020 chr11 76891737 76891759 chr3 15056861 15056883 chrX 24592372 24592393 chr11 85780949 85780971 chr3 162472197 162472218 chrX 26644776 26644797 chr11 88640473 88640495 chr3 192144038 192144059 chrX 55695885 55695906 chr11 92366842 92366863 chr3 193972967 193972988 chrX 6695896 6695917 chr12 104031167 104031188 chr3 31318637 31318658 chrX 97311438 97311459 chr12 131154018 131154039 chr3 33928700 33928721 chrX 9977092 9977114 chr12 131507086 131507107 chr3 66347945 66347967 chrY 11353457 11353478 chr12 22210480 22210502 chr4 172137983 172138005 chr11 9031037 9031063 chr12 2412249 2412270 chr4 37832335 37832356 chr2 135142584 135142611 chr12 37569101 37569123 chr4 53708420 53708441 chr2 166281710 166281737 chr12 50209171 50209193 chr5 133278354 133278375 chr2 166286555 166286581 chr12 79672940 79672961 chr5 155337749 155337770 chr2 166286555 166286582 chr12 93641177 93641198 chr5 39782986 39783009 chr4 139864810 139864837 chr12 95672692 95672713 chr5 67714762 67714784 chr5 163173454 163173480 chr13 21222393 21222415 chr6 35730038 35730059 chr17 63959375 63959402 chr13 24651496 24651519 chr6 44811459 44811480 chr2 166278250 166278277 chr13 29651059 29651081 chr7 149925597 149925618 chr4 46082495 46082522 chr13 84115549 84115570 chr7 152767248 152767269 chr2 166284785 166284812 chr14 34994321 34994343 chr7 156408013 156408034 chr5 120440047 120440073 chr14 75554232 75554254 chr7 28741171 28741192 chrX 148562427 148562453 chr15 36211196 36211217 chr8 29989405 29989427 chr19 13426131 13426157 chr15 62122910 62122931 chr8 34718643 34718664 chr2 166284792 166284819 chr15 76793946 76793968 chr8 43134646 43134667 chr12 123248645 123248671 chr15 98022583 98022605 chr8 752973 752994 chr2 166284621 166284647 chr16 17114450 17114472 chr9 74554912 74554934 chr2 166284621 166284648 chr16 2398065 2398087 chr9 81540528 81540550 chr14 38705215 38705242 chr16 29733409 29733431 chrX 152649787 152649810 chr2 166293356 166293383 chr16 56488215 56488237 chr1 108020984 108021006 chr2 165313740 165313766 chr16 81033918 81033940 chr1 157607538 157607560 chr2 166293225 166293252 chr17 22325036 22325058 chr1 51964038 51964059 chr11 95324148 95324174 chr17 27938284 27938305 chr1 75514790 75514811 chr12 107418063 107418089 chr17 28384097 28384119 chr10 48281408 48281430 chr12 51699625 51699652 chr17 45270070 45270092 chr11 116241444 116241465 chr13 105592802 105592828 chr17 56133866 56133888 chr11 30474472 30474493 chr2 165162747 165162773 chr18 12282949 12282971 chr12 129136215 129136236 chr2 165310378 165310404 chr18 38388400 38388422 chr13 22491824 22491845 chr2 166051907 166051933 chr19 36769800 36769822 chr13 48042239 48042260 chr2 166303220 166303247 chr19 57588377 57588398 chr13 76397102 76397123 chr2 217662908 217662934 chr2 162152161 162152184 chr14 72859229 72859251 chr2 226368493 226368519 chr2 23872600 23872621 chr14 87684513 87684536 chr6 68609343 68609369 chr2 239298584 239298606 chr15 39766578 39766601 chr7 30750602 30750628 chr2 46148451 46148472 chr16 1615112 1615133 chr2 166305804 166305831 chr2 88190010 88190032 chr16 48745003 48745024 chr3 38771291 38771318 chr20 25843288 25843310 chr18 57051391 57051412 chr3 38752265 38752292 chr20 26012592 26012614 chr19 51986606 51986627 chr7 10755206 10755234 chr20 34453128 34453149 chr19 52276544 52276565 chrX 133006058 133006084 chr21 42555302 42555323 chr2 130730007 130730028 chr3 38752213 38752240 chr3 23131297 23131319 chr2 166286321 166286343 chr3 38752415 38752442 chr3 38793741 38793763 chr2 184148698 184148721 chr8 28147462 28147488 chr3 39209399 39209421 chr2 44473938 44473960 chr3 38793739 38793766 chr3 43298469 43298491 chr2 69785973 69785994 chr3 38793953 38793980 chr4 110374339 110374361 chr3 100151370 100151392 chr3 38750104 38750131 chr4 137160917 137160939 chr3 172830944 172830967 chr11 133729424 133729450 chr4 138259457 138259478 chr3 26459710 26459731 chr3 38771273 38771300 chr4 150215252 150215274 chr3 38378001 38378022 chr10 112503420 112503446 chr4 17531757 17531779 chr4 69868253 69868274 chr3 38757064 38757091 chr5 14120019 14120040 chr4 99790429 99790450 chr4 175998006 175998032 chr5 141309428 141309450 chr5 101634255 101634276 chr4 80646320 80646347 chr5 16904562 16904584 chr5 135085796 135085817 chrX 13780722 13780748 chr5 28146632 28146653 chr5 173714270 173714292 chr1 105835449 105835475 chr6 104556941 104556963 chr6 149482923 149482944 chr16 59275323 59275349 chr6 10698822 10698843 chr6 169223323 169223344 chr3 38739609 38739636 chr6 157813372 157813395 chr6 73397278 73397299 chr4 96421786 96421812 chr6 30975969 30975991 chr7 139412916 139412937 chr7 14395432 14395458 chr6 4531543 4531565 chr7 5354372 5354393 chr13 45392357 45392383 chr6 5716528 5716550 chr7 7420448 7420469 chr15 80242156 80242184 chr6 73055476 73055497 chr8 10154295 10154317 chr3 38739575 38739602

Results

Screening of SpCas9 and SaCas9 gRNAs Targeted to SCN9A and SCN10A in iPSCs

Following two rounds of gRNA screening in iPSCs and sequencing analysis, the mean average cutting efficiency was calculated based on four replicates for each sample looking at total percentage of insertions and deletions (indels) at the predicted cut site of each gRNA. For the purposes of knocking out the SCN9A and SCN10A genes, the average percentage of indels that would result in a frameshift mutation was also calculated. Guide RNAs were ranked on both mean total indel percentage and mean frameshift-causing indel percentage. Guide RNAs are listed in rank order based on mean frameshift-causing indel percentages and a scrambled non-targeting gRNA as well as untreated cells were included as negative controls (Tables 8-11).

TABLE 8 Mean total indel percentage and mean frameshift-causing indel percentages generated by SpCas9 gRNAs targeting SCN9A in iPSCs Mean Rank frameshift- Rank based on Mean total causing based on frameshift- Target Target indel indel total causing gRNA name gene exon Cas9 percentage percentage indels indels Scn9a_Sp_Exon_11_T9 SCN9A 11 SpCas9 81.86% 76.55% 3 1 Scn9a_Sp_Exon_12_T25 SCN9A 12 SpCas9 80.50% 76.05% 4 2 Scn9a_Sp_Exon_11_T6 SCN9A 11 SpCas9 78.51% 74.56% 5 3 Scn9a_Sp_Exon_11_T11 SCN9A 11 SpCas9 83.29% 73.18% 1 4 Scn9a_Sp_Exon_12_T20 SCN9A 12 SpCas9 81.94% 73.08% 2 5 Scn9a_Sp_Exon_11_T14 SCN9A 11 SpCas9 76.90% 72.51% 8 6 Scn9a_Sp_Exon_11_T13 SCN9A 11 SpCas9 78.01% 70.14% 7 7 Scn9a_Sp_Exon_14_T1 SCN9A 14 SpCas9 75.27% 69.46% 9 8 Scn9a_Sp_Exon_7_T5 SCN9A 7 SpCas9 74.45% 69.19% 11 9 Scn9a_Sp_Exon_2_T10 SCN9A 2 SpCas9 73.68% 69.08% 12 10 Scn9a_Sp_Exon_12_T11 SCN9A 12 SpCas9 71.34% 69.01% 16 11 Scn9a_Sp_Exon_12_T17 SCN9A 12 SpCas9 75.20% 68.85% 10 12 Scn9a_Sp_Exon_12_T12 SCN9A 12 SpCas9 70.60% 67.13% 17 13 Scn9a_Sp_Exon_9_T10 SCN9A 9 SpCas9 70.41% 66.70% 18 14 Scn9a_Sp_Exon_11_T2 SCN9A 11 SpCas9 72.32% 64.90% 13 15 Scn9a_Sp_Exon_15_T1 SCN9A 15 SpCas9 70.07% 64.78% 19 16 Scn9a_Sp_Exon_9_T14 SCN9A 9 SpCas9 66.06% 60.75% 27 17 Scn9a_Sp_Exon 12_T28 SCN9A 12 SpCas9 68.39% 60.72% 20 18 Scn9a_Sp_Exon_12_T13 SCN9A 12 SpCas9 64.19% 60.35% 31 19 Scn9a_Sp_Exon_11_T5 SCN9A 11 SpCas9 71.86% 59.88% 15 20 Scn9a_Sp_Exon_12_T24 SCN9A 12 SpCas9 68.22% 58.77% 22 21 Scn9a_Sp_Exon_2_T2 SCN9A 2 SpCas9 62.59% 58.70% 35 22 Scn9a_Sp_Exon_12_T21 SCN9A 12 SpCas9 78.19% 58.69% 6 23 Scn9a_Sp_Exon 12_T27 SCN9A 12 SpCas9 67.28% 58.34% 25 24 Scn9a_Sp_Exon_9_T11 SCN9A 9 SpCas9 63.22% 57.35% 34 25 Scn9a_Sp_Exon_12_T1 SCN9A 12 SpCas9 61.57% 57.18% 37 26 Scn9a_Sp_Exon_12_T23 SCN9A 12 SpCas9 64.98% 56.37% 29 27 Scn9a_Sp_Exon_14_T3 SCN9A 14 SpCas9 68.34% 55.35% 21 28 Scn9a_Sp_Exon_10_T2 SCN9A 10 SpCas9 64.20% 54.01% 30 29 Scn9a_Sp_Exon_12_T4 SCN9A 12 SpCas9 61.66% 53.77% 36 30 Scn9a_Sp_Exon_12_T8 SCN9A 12 SpCas9 59.99% 53.24% 41 31 Scn9a_Sp_Exon_11_T10 SCN9A 11 SpCas9 59.73% 52.88% 42 32 Scn9a_Sp_Exon_2_T14 SCN9A 2 SpCas9 63.45% 52.60% 32 33 Scn9a_Sp_Exon_5_T3 SCN9A 5 SpCas9 56.95% 51.72% 49 34 Scn9a_Sp_Exon_12_T2 SCN9A 12 SpCas9 57.05% 51.39% 48 35 Scn9a_Sp_Exon_11_T15 SCN9A 11 SpCas9 67.51% 51.11% 24 36 Scn9a_Sp_Exon_11_T3 SCN9A 11 SpCas9 54.46% 50.05% 54 37 Scn9a_Sp_Exon_9_T6 SCN9A 9 SpCas9 63.44% 49.88% 33 38 Scn9a_Sp_Exon_9_T5 SCN9A 9 SpCas9 53.89% 49.71% 56 39 Scn9a_Sp_Exon_7_T2 SCN9A 7 SpCas9 54.86% 47.44% 52 40 Scn9a_Sp_Exon_2_T4 SCN9A 2 SpCas9 57.70% 46.83% 47 41 Scn9a_Sp_Exon_2_T1 SCN9A 2 SpCas9 66.37% 45.78% 26 42 Scn9a_Sp_Exon_9_T2 SCN9A 9 SpCas9 58.26% 45.73% 46 43 Scn9a_Sp_Exon_4_T5 SCN9A 4 SpCas9 49.69% 45.71% 62 44 Scn9a_Sp_Exon_7_T6 SCN9A 7 SpCas9 60.22% 45.52% 40 45 Scn9a_Sp_Exon_12_T10 SCN9A 12 SpCas9 53.94% 45.27% 55 46 Scn9a_Sp_Exon_4_T3 SCN9A 4 SpCas9 50.92% 45.04% 58 47 Scn9a_Sp_Exon_11_T12 SCN9A 11 SpCas9 59.05% 43.60% 45 48 Scn9a_Sp_Exon_9_T13 SCN9A 9 SpCas9 60.77% 43.49% 39 49 Scn9a_Sp_Exon_11_T7 SCN9A 11 SpCas9 50.76% 43.49% 59 50 Scn9a_Sp_Exon_15_T2 SCN9A 15 SpCas9 59.54% 43.34% 44 51 Scn9a_Sp_Exon_12_T16 SCN9A 12 SpCas9 61.29% 42.92% 38 52 Scn9a_Sp_Exon_12_T19 SCN9A 12 SpCas9 59.59% 42.45% 43 53 Scn9a_Sp_Exon_2_T15 SCN9A 2 SpCas9 44.47% 41.45% 67 54 Scn9a_Sp_Exon_14_T2 SCN9A 14 SpCas9 46.19% 40.97% 64 55 Scn9a_Sp_Exon_2_T13 SCN9A 2 SpCas9 55.38% 40.79% 51 56 Scn9a_Sp_Exon_9_T12 SCN9A 9 SpCas9 44.16% 39.70% 68 57 Scn9a_Sp_Exon_9_T4 SCN9A 9 SpCas9 50.13% 39.54% 61 58 Scn9a_Sp_Exon_2_T11 SCN9A 2 SpCas9 53.70% 39.17% 57 59 Scn9a_Sp_Exon_10_T5 SCN9A 10 SpCas9 55.73% 38.73% 50 60 Scn9a_Sp_Exon_5_T1 SCN9A 5 SpCas9 66.04% 37.17% 28 61 Scn9a_Sp_Exon 12_T7 SCN9A 12 SpCas9 72.23% 37.12% 14 62 Scn9a_Sp_Exon_7_T1 SCN9A 7 SpCas9 50.50% 37.01% 60 63 Scn9a_Sp_Exon_15_T3 SCN9A 15 SpCas9 54.50% 36.77% 53 64 Scn9a_Sp_Exon_12_T3 SCN9A 12 SpCas9 42.40% 35.16% 71 65 Scn9a_Sp_Exon_7_T3 SCN9A 7 SpCas9 46.33% 34.84% 63 66 Scn9a_Sp_Exon_9_T9 SCN9A 9 SpCas9 38.80% 34.59% 77 67 Scn9a_Sp_Exon_5_T2 SCN9A 5 SpCas9 39.80% 34.24% 74 68 Scn9a_Sp_Exon_2_T9 SCN9A 2 SpCas9 45.47% 33.41% 65 69 Scn9a_Sp_Exon 12_T9 SCN9A 12 SpCas9 38.68% 33.01% 79 70 Scn9a_Sp_Exon_12_T5 SCN9A 12 SpCas9 33.67% 31.96% 82 71 Scn9a_Sp_Exon_14_T5 SCN9A 14 SpCas9 44.55% 31.78% 66 72 Scn9a_Sp_Exon_2_T8 SCN9A 2 SpCas9 38.76% 31.73% 78 73 Scn9a_Sp_Exon_9_T15 SCN9A 9 SpCas9 39.98% 31.56% 73 74 Scn9a_Sp_Exon 12_T14 SCN9A 12 SpCas9 33.90% 31.22% 81 75 Scn9a_Sp_Exon_2_T12 SCN9A 2 SpCas9 39.76% 31.02% 75 76 Scn9a_Sp_Exon 12_T18 SCN9A 12 SpCas9 68.21% 30.76% 23 77 Scn9a_Sp_Exon 12_T15 SCN9A 12 SpCas9 39.07% 30.33% 76 78 Scn9a_Sp_Exon_12_T26 SCN9A 12 SpCas9 43.78% 30.13% 70 79 Scn9a_Sp_Exon_5_T4 SCN9A 5 SpCas9 38.57% 27.51% 80 80 Scn9a_Sp_Exon_12_T6 SCN9A 12 SpCas9 31.83% 26.92% 84 81 Scn9a_Sp_Exon_9_T1 SCN9A 9 SpCas9 40.20% 25.44% 72 82 Scn9a_Sp_Exon_2_T7 SCN9A 2 SpCas9 33.06% 25.35% 83 83 Scn9a_Sp_Exon_4_T7 SCN9A 4 SpCas9 26.35% 24.78% 88 84 Scn9a_Sp_Exon_4_T1 SCN9A 4 SpCas9 28.34% 23.77% 86 85 Scn9a_Sp_Exon_2_T3 SCN9A 2 SpCas9 44.01% 23.46% 69 86 Scn9a_Sp_Exon_12_T22 SCN9A 12 SpCas9 24.59% 22.80% 92 87 Scn9a_Sp_Exon_14_T4 SCN9A 14 SpCas9 27.07% 22.67% 87 88 Scn9a_Sp_Exon_4_T2 SCN9A 4 SpCas9 25.94% 22.12% 89 89 Scn9a_Sp_Exon_4_T4 SCN9A 4 SpCas9 24.59% 19.81% 93 90 Scn9a_Sp_Exon_2_T5 SCN9A 2 SpCas9 21.60% 19.28% 95 91 Scn9a_Sp_Exon_9_T3 SCN9A 9 SpCas9 24.82% 18.98% 90 92 Scn9a_Sp_Exon_9_T8 SCN9A 9 SpCas9 31.39% 18.79% 85 93 Scn9a_Sp_Exon_10_T4 SCN9A 10 SpCas9 22.68% 16.55% 94 94 Scn9a_Sp_Exon_9_T7 SCN9A 9 SpCas9 24.72% 15.94% 91 95 Scn9a_Sp_Exon_14_T6 SCN9A 14 SpCas9 17.72% 12.91% 96 96 Scn9a_Sp_Exon_4_T6 SCN9A 4 SpCas9 9.53% 7.42% 97 97 Scn9a_Sp_Exon_2_T6 SCN9A 2 SpCas9 7.09% 5.35% 98 98 Scn9a_Sp_Exon_10_T1 SCN9A 10 SpCas9 1.29% 1.22% 99 99 Scrambled control N/A N/A SpCas9 0.27% 0.23% N/A N/A Untreated N/A N/A N/A 0.18% 0.16% N/A N/A

TABLE 9 Mean total indel percentage and mean frameshift-causing indel percentages generated by SaCas9 gRNAs targeting SCN9A in iPSCs Mean Rank frameshift- Rank based on Mean total causing based on frameshift- Target Target indel indel total causing gRNA name gene exon Cas9 percentage percentage indels indels Scn9a_Sa_Exon_11_T3 SCN9A 11 SaCas9 55.50% 49.49% 2 1 Scn9a_Sa_Exon_12_T2 SCN9A 12 SaCas9 61.96% 49.44% 1 2 Scn9a_Sa_Exon_5_T6 SCN9A 5 SaCas9 54.10% 42.37% 3 3 Scn9a_Sa_Exon_9_T3 SCN9A 9 SaCas9 48.41% 34.60% 4 4 Scn9a_Sa_Exon_13_T3 SCN9A 13 SaCas9 42.29% 33.08% 5 5 Scn9a_Sa_Exon_12_T1 SCN9A 12 SaCas9 33.66% 28.42% 7 6 Scn9a_Sa_Exon_7_T1 SCN9A 7 SaCas9 30.86% 25.39% 8 7 Scn9a_Sa_Exon_9_T2 SCN9A 9 SaCas9 27.99% 24.43% 11 8 Scn9a_Sa_Exon_15_T3 SCN9A 15 SaCas9 28.33% 23.51% 10 9 Scn9a_Sa_Exon_9_T1 SCN9A 9 SaCas9 41.10% 21.52% 6 10 Scn9a_Sa_Exon_12_T3 SCN9A 12 SaCas9 24.81% 20.49% 16 11 Scn9a_Sa_Exon_7_T5 SCN9A 7 SaCas9 25.26% 20.32% 15 12 Scn9a_Sa_Exon_14_T1 SCN9A 14 SaCas9 20.70% 19.06% 20 13 Scn9a_Sa_Exon_2_T2 SCN9A 2 SaCas9 23.85% 18.85% 18 14 Scn9a_Sa_Exon_11_T1 SCN9A 11 SaCas9 26.58% 17.84% 14 15 Scn9a_Sa_Exon_8_T2 SCN9A 8 SaCas9 27.04% 16.09% 13 16 Scn9a_Sa_Exon_9_T4 SCN9A 9 SaCas9 21.90% 15.56% 19 17 Scn9a_Sa_Exon_15_T6 SCN9A 15 SaCas9 19.09% 15.54% 22 18 Scn9a_Sa_Exon_12_T4 SCN9A 12 SaCas9 28.51% 14.30% 9 19 Scn9a_Sa_Exon_5_T1 SCN9A 5 SaCas9 17.45% 14.16% 23 20 Scn9a_Sa_Exon_5_T3 SCN9A 5 SaCas9 16.87% 13.67% 24 21 Scn9a_Sa_Exon_11_T4 SCN9A 11 SaCas9 14.96% 12.34% 26 22 Scn9a_Sa_Exon_5_T2 SCN9A 5 SaCas9 20.59% 10.46% 21 23 Scn9a_Sa_Exon_7_T6 SCN9A 7 SaCas9 12.55% 9.60% 27 24 Scn9a_Sa_Exon_11_T6 SCN9A 11 SaCas9 27.75% 9.21% 12 25 Scn9a_Sa_Exon_12_T5 SCN9A 12 SaCas9 16.55% 8.77% 25 26 Scn9a_Sa_Exon_4_T4 SCN9A 4 SaCas9 9.89% 8.22% 30 27 Scn9a_Sa_Exon_11_T2 SCN9A 11 SaCas9 24.15% 6.75% 17 28 Scn9a_Sa_Exon_13_T1 SCN9A 13 SaCas9 9.03% 6.63% 32 29 Scn9a_Sa_Exon_10_T2 SCN9A 10 SaCas9 12.12% 6.59% 29 30 Scn9a_Sa_Exon_10_T1 SCN9A 10 SaCas9 8.74% 6.55% 33 31 Scn9a_Sa_Exon_2_T1 SCN9A 2 SaCas9 9.69% 6.54% 31 32 Scn9a_Sa_Exon_5_T4 SCN9A 5 SaCas9 12.16% 6.43% 28 33 Scn9a_Sa_Exon_4_T7 SCN9A 4 SaCas9 7.54% 6.35% 36 34 Scn9a_Sa_Exon_15_T5 SCN9A 15 SaCas9 8.00% 5.84% 35 35 Scn9a_Sa_Exon_5_T5 SCN9A 5 SaCas9 7.19% 5.07% 37 36 Scn9a_Sa_Exon_14_T2 SCN9A 14 SaCas9 6.97% 4.96% 39 37 Scn9a_Sa_Exon_6_T5 SCN9A 6 SaCas9 5.78% 4.90% 42 38 Scn9a_Sa_Exon_15_T7 SCN9A 15 SaCas9 8.29% 4.68% 34 39 Scn9a_Sa_Exon_13_T4 SCN9A 13 SaCas9 5.71% 4.33% 44 40 Scn9a_Sa_Exon_5_T7 SCN9A 5 SaCas9 6.54% 4.33% 40 41 Scn9a_Sa_Exon_14_T6 SCN9A 14 SaCas9 5.88% 4.17% 41 42 Scn9a_Sa_Exon_4_T6 SCN9A 4 SaCas9 7.13% 4.00% 38 43 Scn9a_Sa_Exon_13_T2 SCN9A 13 SaCas9 4.50% 3.54% 46 44 Scn9a_Sa_Exon_4_T5 SCN9A 4 SaCas9 5.75% 3.42% 43 45 Scn9a_Sa_Exon_10_T3 SCN9A 10 SaCas9 4.96% 3.34% 45 46 Scn9a_Sa_Exon_8_T1 SCN9A 8 SaCas9 3.76% 3.00% 48 47 Scn9a_Sa_Exon_7_T4 SCN9A 7 SaCas9 4.43% 2.72% 47 48 Scn9a_Sa_Exon_15_T4 SCN9A 15 SaCas9 3.31% 2.53% 49 49 Scn9a_Sa_Exon_11_T5 SCN9A 11 SaCas9 1.53% 1.28% 51 50 Scn9a_Sa_Exon_15_T1 SCN9A 15 SaCas9 1.69% 1.17% 50 51 Scn9a_Sa_Exon_3_T1 SCN9A 3 SaCas9 1.40% 1.05% 52 52 Scn9a_Sa_Exon_15_T2 SCN9A 15 SaCas9 1.27% 0.83% 53 53 Scn9a_Sa_Exon_2_T3 SCN9A 2 SaCas9 1.18% 0.79% 54 54 Scn9a_Sa_Exon_14_T7 SCN9A 14 SaCas9 0.54% 0.48% 56 55 Scn9a_Sa_Exon_4_T1 SCN9A 4 SaCas9 0.40% 0.40% 57 56 Scn9a_Sa_Exon_6_T2 SCN9A 6 SaCas9 0.27% 0.24% 60 57 Scn9a_Sa_Exon_7_T7 SCN9A 7 SaCas9 0.26% 0.22% 61 58 Scn9a_Sa_Exon_7_T2 SCN9A 7 SaCas9 0.27% 0.22% 59 59 Scn9a_Sa_Exon_7_T8 SCN9A 7 SaCas9 0.22% 0.21% 62 60 Scn9a_Sa_Exon_14_T4 SCN9A 14 SaCas9 0.96% 0.20% 55 61 Scn9a_Sa_Exon_14_T8 SCN9A 14 SaCas9 0.31% 0.18% 58 62 Scn9a_Sa_Exon_14_T10 SCN9A 14 SaCas9 0.16% 0.15% 64 63 Scn9a_Sa_Exon_14_T3 SCN9A 14 SaCas9 0.17% 0.14% 63 64 Scn9a_Sa_Exon_2_T4 SCN9A 2 SaCas9 0.15% 0.10% 65 65 Scn9a_Sa_Exon_8_T3 SCN9A 8 SaCas9 0.09% 0.09% 66 66 Scn9a_Sa_Exon_14_T5 SCN9A 14 SaCas9 0.04% 0.04% 67 67 Scn9a_Sa_Exon_14_T9 SCN9A 14 SaCas9 0.02% 0.02% 68 68 Scrambled control N/A N/A SaCas9 0.27% 0.23% N/A N/A Untreated N/A N/A N/A 0.18% 0.16% N/A N/A

TABLE 10 Mean total indel percentage and mean frameshift-causing indel percentages generated by SpCas9 gRNAs targeting SCN10A in iPSCs Mean Rank frameshift- Rank based on Mean total causing based on frameshift- Target Target indel indel total causing gRNA name gene exon Cas9 percentage percentage indels indels Scn10a_Sp_Exon_1_T15 SCN10A 1 SpCas9 75.77% 73.46% 1 1 Scn10a_Sp_Exon_9_T2 SCN10A 9 SpCas9 70.79% 67.64% 5 2 Scn10a_Sp_Exon_14_T3 SCN10A 14 SpCas9 75.21% 67.26% 2 3 Scn10a_Sp_Exon_10_T7 SCN10A 10 SpCas9 69.16% 66.79% 7 4 Scn10a_Sp_Exon_7_T6 SCN10A 7 SpCas9 69.47% 65.31% 6 5 Scn10a_Sp_Exon_1_T30 SCN10A 1 SpCas9 68.89% 63.21% 8 6 Scn10a_Sp_Exon_11_T10 SCN10A 11 SpCas9 72.63% 62.38% 3 7 Scn10a_Sp_Exon_1_T17 SCN10A 1 SpCas9 64.42% 61.62% 18 8 Scn10a_Sp_Exon_4_T2 SCN10A 4 SpCas9 66.31% 61.10% 12 9 Scn10a_Sp_Exon_10_T3 SCN10A 10 SpCas9 66.83% 60.57% 9 10 Scn10a_Sp_Exon_8_T3 SCN10A 8 SpCas9 66.35% 59.76% 11 11 Scn10a_Sp_Exon_1_T18 SCN10A 1 SpCas9 65.19% 59.51% 15 12 Scn10a_Sp_Exon_1_T16 SCN10A 1 SpCas9 66.52% 59.43% 10 13 Scn10a_Sp_Exon_11_T24 SCN10A 11 SpCas9 62.43% 58.84% 24 14 Scn10a_Sp_Exon_12_T3 SCN10A 12 SpCas9 64.78% 58.60% 17 15 Scn10a_Sp_Exon_8_T8 SCN10A 8 SpCas9 65.43% 58.01% 14 16 Scn10a_Sp_Exon_4_T4 SCN10A 4 SpCas9 61.86% 57.86% 25 17 Scn10a_Sp_Exon_1_T14 SCN10A 1 SpCas9 63.17% 57.59% 22 18 Scn10a_Sp_Exon_14_T1 SCN10A 14 SpCas9 71.62% 56.93% 4 19 Scn10a_Sp_Exon_13_T4 SCN10A 13 SpCas9 61.76% 55.63% 26 20 Scn10a_Sp_Exon_8_T10 SCN10A 8 SpCas9 60.22% 54.64% 33 21 Scn10a_Sp_Exon_7_T2 SCN10A 7 SpCas9 60.19% 54.34% 34 22 Scn10a_Sp_Exon_13_T5 SCN10A 13 SpCas9 57.14% 53.58% 44 23 Scn10a_Sp_Exon_10_T2 SCN10A 10 SpCas9 58.03% 53.29% 38 24 Scn10a_Sp_Exon_7_T7 SCN10A 7 SpCas9 56.45% 52.36% 48 25 Scn10a_Sp_Exon_13_T17 SCN10A 13 SpCas9 56.62% 51.99% 47 26 Scn10a_Sp_Exon_9_T14 SCN10A 9 SpCas9 63.41% 51.82% 20 27 Scn10a_Sp_Exon_1_T25 SCN10A 1 SpCas9 60.51% 51.36% 32 28 Scn10a_Sp_Exon_12_T6 SCN10A 12 SpCas9 56.11% 50.72% 50 29 Scn10a_Sp_Exon_12_T8 SCN10A 12 SpCas9 57.60% 50.69% 41 30 Scn10a_Sp_Exon_2_T3 SCN10A 2 SpCas9 63.38% 50.51% 21 31 Scn10a_Sp_Exon_13_T18 SCN10A 13 SpCas9 57.69% 49.95% 40 32 Scn10a_Sp_Exon_11_T14 SCN10A 11 SpCas9 59.24% 49.80% 36 33 Scn10a_Sp_Exon_11_T19 SCN10A 11 SpCas9 53.70% 49.80% 67 34 Scn10a_Sp_Exon_13_T9 SCN10A 13 SpCas9 65.73% 49.73% 13 35 Scn10a_Sp_Exon_8_T1 SCN10A 8 SpCas9 56.68% 49.55% 46 36 Scn10a_Sp_Exon_1_T10 SCN10A 1 SpCas9 54.89% 49.53% 60 37 Scn10a_Sp_Exon_2_T7 SCN10A 2 SpCas9 60.11% 49.29% 35 38 Scn10a_Sp_Exon_11_T25 SCN10A 11 SpCas9 62.96% 49.26% 23 39 Scn10a_Sp_Exon_1_T3 SCN10A 1 SpCas9 55.92% 48.35% 51 40 Scn10a_Sp_Exon_14_T2 SCN10A 14 SpCas9 57.55% 48.19% 42 41 Scn10a_Sp_Exon_10_T10 SCN10A 10 SpCas9 52.18% 48.08% 77 42 Scn10a_Sp_Exon_13_T13 SCN10A 13 SpCas9 54.65% 47.73% 61 43 Scn10a_Sp_Exon_11_T28 SCN10A 11 SpCas9 60.69% 47.45% 31 44 Scn10a_Sp_Exon_5_T4 SCN10A 5 SpCas9 50.66% 47.09% 87 45 Scn10a_Sp_Exon_9_T3 SCN10A 9 SpCas9 51.41% 46.55% 81 46 Scn10a_Sp_Exon_2_T5 SCN10A 2 SpCas9 50.44% 46.45% 88 47 Scn10a_Sp_Exon_5_T1 SCN10A 5 SpCas9 52.12% 46.30% 78 48 Scn10a_Sp_Exon_11_T16 SCN10A 11 SpCas9 60.99% 45.94% 30 49 Scn10a_Sp_Exon_11_T18 SCN10A 11 SpCas9 61.41% 45.75% 28 50 Scn10a_Sp_Exon_4_T1 SCN10A 4 SpCas9 54.98% 45.45% 58 51 Scn10a_Sp_Exon_12_T5 SCN10A 12 SpCas9 61.45% 45.41% 27 52 Scn10a_Sp_Exon_2_T8 SCN10A 2 SpCas9 54.37% 45.09% 63 53 Scn10a_Sp_Exon_11_T21 SCN10A 11 SpCas9 55.63% 44.13% 54 54 Scn10a_Sp_Exon_11_T17 SCN10A 11 SpCas9 54.96% 44.09% 59 55 Scn10a_Sp_Exon_11_T15 SCN10A 11 SpCas9 48.65% 44.08% 95 56 Scn10a_Sp_Exon_13_T19 SCN10A 13 SpCas9 51.06% 43.61% 83 57 Scn10a_Sp_Exon_6_T3 SCN10A 6 SpCas9 54.41% 43.58% 62 58 Scn10a_Sp_Exon_10_T1 SCN10A 10 SpCas9 57.92% 43.50% 39 59 Scn10a_Sp_Exon_11_T11 SCN10A 11 SpCas9 55.69% 43.45% 53 60 Scn10a_Sp_Exon_10_T6 SCN10A 10 SpCas9 49.04% 43.14% 93 61 Scn10a_Sp_Exon_12_T7 SCN10A 12 SpCas9 52.96% 43.13% 71 62 Scn10a_Sp_Exon_1_T5 SCN10A 1 SpCas9 48.50% 43.07% 97 63 Scn10a_Sp_Exon_13_T16 SCN10A 13 SpCas9 50.90% 42.78% 86 64 Scn10a_Sp_Exon_9_T13 SCN10A 9 SpCas9 64.98% 42.52% 16 65 Scn10a_Sp_Exon_6_T7 SCN10A 6 SpCas9 58.51% 42.38% 37 66 Scn10a_Sp_Exon_7_T4 SCN10A 7 SpCas9 52.52% 42.37% 75 67 Scn10a_Sp_Exon_9_T4 SCN10A 9 SpCas9 53.88% 42.24% 66 68 Scn10a_Sp_Exon_9_T9 SCN10A 9 SpCas9 52.53% 42.19% 74 69 Scn10a_Sp_Exon_10_T9 SCN10A 10 SpCas9 47.84% 42.05% 100 70 Scn10a_Sp_Exon_8_T2 SCN10A 8 SpCas9 47.46% 41.70% 102 71 Scn10a_Sp_Exon_1_T9 SCN10A 1 SpCas9 50.32% 41.44% 90 72 Scn10a_Sp_Exon_13_T15 SCN10A 13 SpCas9 55.09% 41.15% 57 73 Scn10a_Sp_Exon_1_T26 SCN10A 1 SpCas9 48.17% 40.83% 99 74 Scn10a_Sp_Exon_6_T8 SCN10A 6 SpCas9 50.36% 40.76% 89 75 Scn10a_Sp_Exon_1_T4 SCN10A 1 SpCas9 51.44% 40.43% 80 76 Scn10a_Sp_Exon_11_T22 SCN10A 11 SpCas9 52.25% 40.22% 76 77 Scn10a_Sp_Exon_1_T8 SCN10A 1 SpCas9 50.94% 40.09% 85 78 Scn10a_Sp_Exon_1_T28 SCN10A 1 SpCas9 55.88% 40.04% 52 79 Scn10a_Sp_Exon_1_T23 SCN10A 1 SpCas9 48.87% 39.94% 94 80 Scn10a_Sp_Exon_12_T14 SCN10A 12 SpCas9 53.67% 39.85% 68 81 Scn10a_Sp_Exon_10_T5 SCN10A 10 SpCas9 45.47% 39.07% 104 82 Scn10a_Sp_Exon_8_T9 SCN10A 8 SpCas9 53.65% 38.83% 69 83 Scn10a_Sp_Exon_10_T4 SCN10A 10 SpCas9 53.90% 38.71% 65 84 Scn10a_Sp_Exon_2_T6 SCN10A 2 SpCas9 45.38% 38.38% 105 85 Scn10a_Sp_Exon_8_T6 SCN10A 8 SpCas9 57.30% 38.26% 43 86 Scn10a_Sp_Exon_12_T9 SCN10A 12 SpCas9 42.87% 38.17% 107 87 Scn10a_Sp_Exon_2_T10 SCN10A 2 SpCas9 47.78% 37.61% 101 88 Scn10a_Sp_Exon_12_T1 SCN10A 12 SpCas9 49.42% 36.83% 91 89 Scn10a_Sp_Exon_14_T4 SCN10A 14 SpCas9 61.35% 35.92% 29 90 Scn10a_Sp_Exon_2_T9 SCN10A 2 SpCas9 53.15% 35.54% 70 91 Scn10a_Sp_Exon_1_T19 SCN10A 1 SpCas9 48.57% 35.36% 96 92 Scn10a_Sp_Exon_13_T19 SCN10A 11 SpCas9 42.69% 34.85% 108 93 Scn10a_Sp_Exon_11_T20 SCN10A 13 SpCas9 39.77% 34.85% 120 94 Scn10a_Sp_Exon_13_T10 SCN10A 13 SpCas9 40.55% 34.73% 117 95 Scn10a_Sp_Exon_12_T4 SCN10A 12 SpCas9 42.32% 34.32% 111 96 Scn10a_Sp_Exon_11_T29 SCN10A 11 SpCas9 55.45% 34.29% 55 97 Scn10a_Sp_Exon_1_T6 SCN10A 1 SpCas9 41.41% 33.80% 114 98 Scn10a_Sp_Exon_11_T5 SCN10A 11 SpCas9 36.79% 33.61% 127 99 Scn10a_Sp_Exon_8_T4 SCN10A 8 SpCas9 48.33% 33.60% 98 100 Scn10a_Sp_Exon_1_T20 SCN10A 1 SpCas9 40.84% 33.49% 116 101 Scn10a_Sp_Exon_11_T23 SCN10A 11 SpCas9 52.78% 33.30% 73 102 Scn10a_Sp_Exon_6_T4 SCN10A 6 SpCas9 38.65% 33.10% 121 103 Scn10a_Sp_Exon_1_T7 SCN10A 1 SpCas9 44.90% 32.82% 106 104 Scn10a_Sp_Exon_2_T2 SCN10A 2 SpCas9 51.23% 32.78% 82 105 Scn10a_Sp_Exon_1_T24 SCN10A 1 SpCas9 42.10% 32.67% 112 106 Scn10a_Sp_Exon_4_T5 SCN10A 4 SpCas9 56.88% 32.61% 45 107 Scn10a_Sp_Exon_9_T11 SCN10A 9 SpCas9 52.09% 32.18% 79 108 Scn10a_Sp_Exon_9_T1 SCN10A 9 SpCas9 41.37% 32.00% 115 109 Scn10a_Sp_Exon_11_T27 SCN10A 11 SpCas9 55.20% 31.85% 56 110 Scn10a_Sp_Exon_11_T1 SCN10A 11 SpCas9 50.96% 31.76% 84 111 Scn10a_Sp_Exon_11_T30 SCN10A 11 SpCas9 34.52% 31.72% 133 112 Scn10a_Sp_Exon_13_T1 SCN10A 13 SpCas9 37.55% 31.55% 125 113 Scn10a_Sp_Exon_9_T8 SCN10A 9 SpCas9 52.96% 31.47% 72 114 Scn10a_Sp_Exon_4_T3 SCN10A 4 SpCas9 36.31% 31.31% 130 115 Scn10a_Sp_Exon_1_T21 SCN10A 1 SpCas9 42.58% 31.22% 109 116 Scn10a_Sp_Exon_8_T5 SCN10A 8 SpCas9 49.09% 30.85% 92 117 Scn10a_Sp_Exon_13_T12 SCN10A 13 SpCas9 42.38% 30.40% 110 118 Scn10a_Sp_Exon_12_T13 SCN10A 12 SpCas9 35.97% 30.38% 131 119 Scn10a_Sp_Exon_6_T9 SCN10A 6 SpCas9 34.43% 30.28% 134 120 Scn10a_Sp_Exon_11_T4 SCN10A 11 SpCas9 54.02% 30.26% 64 121 Scn10a_Sp_Exon_11_T3 SCN10A 11 SpCas9 38.32% 30.19% 123 122 Scn10a_Sp_Exon_12_T10 SCN10A 12 SpCas9 63.89% 29.99% 19 123 Scn10a_Sp_Exon_7_T3 SCN10A 7 SpCas9 38.44% 29.43% 122 124 Scn10a_Sp_Exon_7_T5 SCN10A 7 SpCas9 35.95% 28.88% 132 125 Scn10a_Sp_Exon_8_T7 SCN10A 8 SpCas9 40.06% 28.67% 118 126 Scn10a_Sp_Exon_1_T11 SCN10A 1 SpCas9 36.94% 28.57% 126 127 Scn10a_Sp_Exon_11_T33 SCN10A 11 SpCas9 36.65% 27.54% 129 128 Scn10a_Sp_Exon_6_T10 SCN10A 6 SpCas9 32.46% 26.95% 137 129 Scn10a_Sp_Exon_11_T12 SCN10A 11 SpCas9 32.19% 26.77% 138 130 Scn10a_Sp_Exon_11_T7 SCN10A 11 SpCas9 29.15% 26.06% 143 131 Scn10a_Sp_Exon_1_T29 SCN10A 1 SpCas9 46.08% 25.89% 103 132 Scn10a_Sp_Exon_10_T13 SCN10A 10 SpCas9 33.89% 25.37% 135 133 Scn10a_Sp_Exon_13_T8 SCN10A 13 SpCas9 37.57% 25.01% 124 134 Scn10a_Sp_Exon_13_T11 SCN10A 13 SpCas9 29.76% 24.56% 142 135 Scn10a_Sp_Exon_5_T2 SCN10A 5 SpCas9 31.74% 24.55% 139 136 Scn10a_Sp_Exon_11_T13 SCN10A 11 SpCas9 36.77% 24.35% 128 137 Scn10a_Sp_Exon_5_T3 SCN10A 5 SpCas9 30.72% 23.38% 140 138 Scn10a_Sp_Exon_12_T12 SCN10A 12 SpCas9 41.69% 22.51% 113 139 Scn10a_Sp_Exon_9_T5 SCN10A 9 SpCas9 56.19% 22.49% 49 140 Scn10a_Sp_Exon_2_T1 SCN10A 2 SpCas9 30.63% 22.14% 141 141 Scn10a_Sp_Exon_11_T26 SCN10A 11 SpCas9 28.87% 22.10% 144 142 Scn10a_Sp_Exon_11_T8 SCN10A 11 SpCas9 39.80% 21.90% 119 143 Scn10a_Sp_Exon_11_T32 SCN10A 11 SpCas9 26.86% 20.23% 148 144 Scn10a_Sp_Exon_13_T14 SCN10A 13 SpCas9 25.54% 20.20% 151 145 Scn10a_Sp_Exon_1_T27 SCN10A 1 SpCas9 23.64% 20.09% 155 146 Scn10a_Sp_Exon_6_T2 SCN10A 6 SpCas9 24.45% 19.92% 153 147 Scn10a_Sp_Exon_13_T21 SCN10A 13 SpCas9 26.89% 19.88% 147 148 Scn10a_Sp_Exon_11_T2 SCN10A 11 SpCas9 33.54% 19.60% 136 149 Scn10a_Sp_Exon_11_T31 SCN10A 11 SpCas9 22.87% 18.35% 156 150 Scn10a_Sp_Exon_11_T20 SCN10A 11 SpCas9 28.68% 17.60% 145 151 Scn10a_Sp_Exon_1_T2 SCN10A 1 SpCas9 21.35% 17.50% 158 152 Scn10a_Sp_Exon_2_T4 SCN10A 2 SpCas9 22.73% 16.87% 157 153 Scn10a_Sp_Exon_14_T5 SCN10A 14 SpCas9 26.01% 16.71% 150 154 Scn10a_Sp_Exon_9_T10 SCN10A 9 SpCas9 24.38% 16.58% 154 155 Scn10a_Sp_Exon_11_T6 SCN10A 11 SpCas9 17.51% 13.60% 160 156 Scn10a_Sp_Exon_9_T6 SCN10A 9 SpCas9 16.03% 12.46% 161 157 Scn10a_Sp_Exon_1_T22 SCN10A 1 SpCas9 25.18% 12.18% 152 158 Scn10a_Sp_Exon_7_T1 SCN10A 7 SpCas9 27.05% 11.69% 146 159 Scn10a_Sp_Exon_12_T2 SCN10A 12 SpCas9 26.39% 11.56% 149 160 Scn10a_Sp_Exon_9_T12 SCN10A 9 SpCas9 18.60% 10.73% 159 161 Scn10a_Sp_Exon_13_T6 SCN10A 13 SpCas9 9.77% 7.31% 163 162 Scn10a_Sp_Exon_1_T12 SCN10A 1 SpCas9 8.22% 6.02% 164 163 Scn10a_Sp_Exon_9_T7 SCN10A 9 SpCas9 12.54% 5.44% 162 164 Scn10a_Sp_Exon_13_T22 SCN10A 13 SpCas9 6.59% 4.29% 165 165 Scn10a_Sp_Exon_11_T34 SCN10A 11 SpCas9 3.63% 2.42% 166 166 Scrambled control N/A N/A SpCas9 0.27% 0.23% N/A N/A Untreated N/A N/A N/A 0.18% 0.16% N/A N/A

TABLE 11 Mean total indel percentage and mean frameshift-causing indel percentages generated by SaCas9 gRNAs targeting SCN10A in iPSCs Mean Rank frameshift- Rank based on Mean total causing based on frameshift- Target Target indel indel total causing gRNA name gene exon Cas9 percentage percentage indels indels Scn10a_Sa_Exon_11_T1 SCN10A 11 SaCas9 44.86% 42.68% 2 1 Scn10a_Sa_Exon_1_T7 SCN10A 1 SaCas9 42.84% 40.36% 4 2 Scn10a_Sa_Exon_8_T5 SCN10A 8 SaCas9 37.46% 35.32% 7 3 Scn10a_Sa_Exon_12_T2 SCN10A 12 SaCas9 43.31% 32.36% 3 4 Scn10a_Sa_Exon_14_T1 SCN10A 14 SaCas9 45.19% 31.27% 1 5 Scn10a_Sa_Exon_14_T2 SCN10A 14 SaCas9 39.18% 27.17% 5 6 Scn10a_Sa_Exon_4_T5 SCN10A 4 SaCas9 33.11% 25.46% 9 7 Scn10a_Sa_Exon_1_T3 SCN10A 1 SaCas9 28.02% 24.67% 13 8 Scn10a_Sa_Exon_11_T2 SCN10A 11 SaCas9 31.11% 23.87% 11 9 Scn10a_Sa_Exon_11_T5 SCN10A 11 SaCas9 29.04% 23.03% 12 10 Scn10a_Sa_Exon_13_T9 SCN10A 13 SaCas9 34.53% 20.42% 8 11 Scn10a_Sa_Exon_3_T1 SCN10A 3 SaCas9 27.38% 19.18% 14 12 Scn10a_Sa_Exon_13_T2 SCN10A 13 SaCas9 31.25% 18.78% 10 13 Scn10a_Sa_Exon_10_T6 SCN10A 10 SaCas9 20.74% 17.76% 20 14 Scn10a_Sa_Exon_11_T10 SCN10A 11 SaCas9 20.36% 17.65% 22 15 Scn10a_Sa_Exon_2_T1 SCN10A 2 SaCas9 19.59% 17.00% 27 16 Scn10a_Sa_Exon_8_T1 SCN10A 8 SaCas9 25.69% 16.98% 16 17 Scn10a_Sa_Exon_4_T7 SCN10A 4 SaCas9 20.06% 16.79% 24 18 Scn10a_Sa_Exon_13_T4 SCN10A 13 SaCas9 19.68% 15.79% 26 19 Scn10a_Sa_Exon_11_T4 SCN10A 11 SaCas9 20.35% 15.76% 23 20 Scn10a_Sa_Exon_1_T6 SCN10A 1 SaCas9 26.38% 15.31% 15 21 Scn10a_Sa_Exon_7_T4 SCN10A 7 SaCas9 22.50% 15.28% 17 22 Scn10a_Sa_Exon_1_T8 SCN10A 1 SaCas9 21.83% 14.52% 18 23 Scn10a_Sa_Exon_11_T9 SCN10A 11 SaCas9 38.57% 13.91% 6 24 Scn10a_Sa_Exon_7_T3 SCN10A 7 SaCas9 20.06% 13.70% 25 25 Scn10a_Sa_Exon_11_T8 SCN10A 11 SaCas9 21.79% 13.40% 19 26 Scn10a_Sa_Exon_5_T2 SCN10A 5 SaCas9 16.95% 13.18% 29 27 Scn10a_Sa_Exon_1_T4 SCN10A 1 SaCas9 18.11% 13.04% 28 28 Scn10a_Sa_Exon_6_T6 SCN10A 6 SaCas9 20.70% 10.65% 21 29 Scn10a_Sa_Exon_1_T5 SCN10A 1 SaCas9 14.58% 10.63% 32 30 Scn10a_Sa_Exon_4_T2 SCN10A 4 SaCas9 13.82% 10.55% 33 31 Scn10a_Sa_Exon_10_T2 SCN10A 10 SaCas9 15.04% 9.94% 31 32 Scn10a_Sa_Exon_10_T7 SCN10A 10 SaCas9 16.61% 8.83% 30 33 Scn10a_Sa_Exon_13_T3 SCN10A 13 SaCas9 12.36% 8.21% 35 34 Scn10a_Sa_Exon_10_T5 SCN10A 10 SaCas9 10.62% 7.87% 37 35 Scn10a_Sa_Exon_10_T3 SCN10A 10 SaCas9 13.45% 7.63% 34 36 Scn10a_Sa_Exon_8_T4 SCN10A 8 SaCas9 7.75% 6.06% 41 37 Scn10a_Sa_Exon_11_T7 SCN10A 11 SaCas9 6.42% 5.61% 46 38 Scn10a_Sa_Exon_7_T2 SCN10A 7 SaCas9 7.54% 5.53% 42 39 Scn10a_Sa_Exon_13_T5 SCN10A 13 SaCas9 5.84% 5.42% 48 40 Scn10a_Sa_Exon_13_T8 SCN10A 13 SaCas9 7.45% 5.16% 43 41 Scn10a_Sa_Exon_3_T2 SCN10A 3 SaCas9 6.18% 5.14% 47 42 Scn10a_Sa_Exon_4_T4 SCN10A 4 SaCas9 10.64% 4.88% 36 43 Scn10a_Sa_Exon_6_T3 SCN10A 6 SaCas9 8.43% 4.83% 39 44 Scn10a_Sa_Exon_10_T8 SCN10A 10 SaCas9 6.74% 4.64% 44 45 Scn10a_Sa_Exon_1_T9 SCN10A 1 SaCas9 5.42% 4.53% 49 46 Scn10a_Sa_Exon_13_T1 SCN10A 13 SaCas9 6.56% 4.34% 45 47 Scn10a_Sa_Exon_6_T8 SCN10A 6 SaCas9 8.25% 3.89% 40 48 Scn10a_Sa_Exon_4_T3 SCN10A 4 SaCas9 4.37% 3.72% 51 49 Scn10a_Sa_Exon_5_T4 SCN10A 5 SaCas9 5.02% 3.69% 50 50 Scn10a_Sa_Exon_6_T5 SCN10A 6 SaCas9 4.27% 3.66% 52 51 Scn10a_Sa_Exon_13_T7 SCN10A 13 SaCas9 9.36% 2.84% 38 52 Scn10a_Sa_Exon_10_T4 SCN10A 10 SaCas9 2.67% 2.40% 54 53 Scn10a_Sa_Exon_5_T1 SCN10A 5 SaCas9 2.76% 2.36% 53 54 Scn10a_Sa_Exon_2_T2 SCN10A 2 SaCas9 2.29% 1.87% 55 55 Scn10a_Sa_Exon_14_T3 SCN10A 14 SaCas9 2.16% 1.62% 57 56 Scn10a_Sa_Exon_3_T3 SCN10A 3 SaCas9 2.17% 1.53% 56 57 Scn10a_Sa_Exon_12_T4 SCN10A 12 SaCas9 2.07% 1.52% 59 58 Scn10a_Sa_Exon_7_T5 SCN10A 7 SaCas9 2.12% 1.42% 58 59 Scn10a_Sa_Exon_6_T1 SCN10A 6 SaCas9 1.69% 1.21% 61 60 Scn10a_Sa_Exon_11_T3 SCN10A 11 SaCas9 1.93% 0.77% 60 61 Scn10a_Sa_Exon_6_T4 SCN10A 6 SaCas9 0.73% 0.53% 64 62 Scn10a_Sa_Exon_9_T1 SCN10A 9 SaCas9 0.79% 0.47% 63 63 Scn10a_Sa_Exon_10_T1 SCN10A 10 SaCas9 0.46% 0.45% 65 64 Scn10a_Sa_Exon_14_T4 SCN10A 14 SaCas9 1.24% 0.33% 62 65 Scn10a_Sa_Exon_9_T3 SCN10A 9 SaCas9 0.28% 0.20% 67 66 Scn10a_Sa_Exon_1_T1 SCN10A 1 SaCas9 0.20% 0.17% 68 67 Scn10a_Sa_Exon_13_T6 SCN10A 13 SaCas9 0.35% 0.17% 66 68 Scn10a_Sa_Exon_12_T1 SCN10A 12 SaCas9 0.18% 0.16% 69 69 Scn10a_Sa_Exon_8_T2 SCN10A 8 SaCas9 0.11% 0.10% 70 70 Scn10a_Sa_Exon_12_T5 SCN10A 12 SaCas9 0.06% 0.06% 71 71 Scn10a_Sa_Exon_8_T3 SCN10A 8 SaCas9 0.03% 0.03% 72 72 Scn10a_Sa_Exon_6_T7 SCN10A 6 SaCas9 0.00% 0.00% 73 73 Scrambled control N/A N/A SaCas9 0.27% 0.23% N/A N/A Untreated N/A N/A N/A 0.18% 0.14% N/A N/A Screening of Top Ranked gRNAs Targeted to SCN9A and SCN10A in iPSCs Stably Expressing SpCas9 or SaCas9, and in iPSC-Derived Sensory Neurons

Based on on-target efficacy in initial gRNA screens in iPSCs, 40 guides were prioritized for further on-target editing studies in additional cell models such as iPSCs stably expressing Cas9 and iPSC-derived sensory neurons (iSNs) (FIGS. 1A-1D). Specifically, ten guides from each of four categories were chosen: 1) ten gRNAs for SpCas9 targeting SCN9A, 2) ten gRNAs for SpCas9 targeting SCN10a, 3) ten gRNAs for SaCas9 targeting SCN9A, and 4) ten gRNAs for SaCas9 targeting SCN10a (Tables 12 and 13).

These 40 prioritized gRNAs were screened in engineered iPSCs stably expressing either SpCas9 or SaCas9. Synthetic gRNAs were electroporated into the corresponding cell line. These 40 gRNAs were already screened for on-target editing efficiency in iSNs. In iSNs, RNP complexes were electroporated into the adherent neuronal cultures for all 40 gRNAs. In addition, the 20 SaCas9 gRNAs were also delivered to iSNs by all-in-one AAV vectors expressing SaCas9 and a gRNA. Genomic DNA was purified from treated cells for sequencing analysis as described in the methods.

In each model, two independent experiments were conducted. The mean average cutting efficiency was calculated based on four replicates for each sample looking at total percentage of insertions and deletions (indels) at the predicted cut site of each gRNA. For the purposes of knocking out the SCN9A and SCN10A genes, the average percentage of indels that would result in a frameshift mutation was also calculated. Guide RNAs were ranked on mean frameshift-causing indel percentage. A summary of the on-target editing efficiencies of these 40 prioritized gRNAs across different cell models can be found in FIGS. 1A-1D, as well as in Tables 12 and 13.

TABLE 12  Summary of on-target editing efficiency of prioritized SpCas9 gRNAs across different cells models % Frameshift Indels SEQ stable iSNs iPSC ID Cas9 (RNP iPSC stable iSN gRNA Name NO: Sequence iPSCs iPSCs electroporation) rank rank rank SCN9A SpCas9 Scn9a Sp Exon 11_T9 1 CAAuuuGGGuGGuACCuGAu 76.55% 78.18% 15.83% 1 7 8 Scn9a Sp Exon 12_T25 2 GCuuCGCCuuGCAGAAAACA 76.05% 80.74% 33.90% 2 4 3 Scn9a Sp Exon 11_T6 3 GCCuAuGCCCuuCGACACCA 74.56% 85.16% 32.52% 3 2 4 Scn9a Sp Exon 11_T14 4 AuAGGCGAGCACAuGAAAAG 72.51% 75.68%  9.20% 4 9 10 Scn9a Sp Exon 11_T13 5 CGGCuGAAuAuACAAGuAuu 70.14% 79.25% 26.04% 5 5 5 Scn9a Sp Exon 14_T1 6 GGAACACCACCCAAuGACuG 69.46% 78.77% 22.14% 6 6 6 Scn9a Sp Exon 7_T5 7 CAGGCCuGAAGACAAuuGuA 69.19% 81.38% 44.42% 7 3 1 Scn9a Sp Exon 2_T10 8 GGAAuGuCCCCAuAGAuGAA 69.08% 86.34% 37.78% 8 1 2 Scn9a Sp Exon 12_T11 9 CCACCAAuGCuGCCGGuGAA 69.01% 77.06% 14.40% 9 8 9 Scn9a Sp Exon 12_T17 10 CAGuCACCACuCAGCAuuCG 68.85% 70.36% 17.25% 10 10 7 SCN10A SpCas9 Scn10a Sp Exon 1_T15 21 GCuCCCCGAuCAGuuCuGCu 73.46% 79.12% 21.33% 1 6 2 Scn10a Sp Exon 9_T2 22 uGuAGuCACCAuGGCGuAuG 67.64% 80.40% 22.50% 2 4 1 Scn10a Sp Exon 14_T3 23 GGAAGCuCCGCAGCACAGAC 67.26% 82.00% 20.91% 3 3 3 Scn10a Sp Exon 10_T7 24 uCCuuACAACCAGCGCAGGA 66.79% 85.44% 20.77% 4 1 4 Scn10a Sp Exon 7_T6 25 ACuuCuGACCCCuuACuGuG 65.31% 78.28% 19.31% 5 7 6 Scn10a Sp Exon 1_T30 26 GAGCuCCCAGCAGAACuGAu 63.21% 71.66% 17.33% 6 9 7 Scn10a Sp Exon 11_T10 27 CCGAGACAuCGACAGCuCCA 62.38% 76.53% 16.60% 7 8 9 Scn10a Sp Exon 1_T17 28 AuCCGuuCuACAGCACACAC 61.62% 83.60% 20.25% 8 2 5 Scn10a Sp Exon 4_T2 29 uCACGuACCuGAGAGAuCCu 61.10% 65.50%  8.80% 9 10 10 Scn10a Sp Exon 10_T3 30 CGCAGGuGCuAGCAGCACuA 60.57% 79.22% 17.04% 10 5 8

TABLE 13  Summary of on-target editing efficiency of prioritized SaCas9 gRNAs across different cells models % Frameshift Indels iSNs iSNs SEQ stable (RNP (AAV iPSC iSN iSN ID Cas9 electro- trans- iPSC stable (RNP) (AAV) gRNA Name NO: Sequence iPSCs iPSCs poration) duction) rank rank rank rank SCN9A SaCas9 Scn9a Sa Exon 11_T3 11 AAGCAGAAuuAuGGGCCuCuCA 49.49% 66.77%  7.87% 14.87% 1 1 4 3 Scn9a Sa Exon 12_T2 12 GCCuuGCAGAAAACAAGGAGCC 49.44% 57.62% 14.87% 23.22% 2 2 1 1 Scn9a Sa Exon 5_T6 13 ACGACAAAAuCCAGCCAGuuCC 42.37% 54.16% 11.97%  3.79% 3 3 3 10 Scn9a Sa Exon 9_T3 14 CuGGGAAAACCuuuACCAACAG 34.60% 48.90% 13.50%  6.20% 4 7 2 8 Scn9a Sa Exon 13_T3 15 uCCCAACCuCAGACAGAGAGCA 33.08% 51.37%  4.33% 13.04% 5 5 7 4 Scn9a Sa Exon 12_T1 16 GAuGuuACuGCuGCGuCGCuCC 28.42% 52.96%  7.57%  9.96% 6 4 5 6 Scn9a Sa Exon 7_T1 17 CAuGAuCCuGACuGuGuuCuGu 25.39% 40.83%  4.22%  5.70% 7 10 8 9 Scn9a Sa Exon 9_T2 18 CuCGuGuGuAGuCAGuGuCCAG 24.43% 51.04%  3.84% 12.52% 8 6 9 5 Scn9a Sa Exon 15_T3 19 AAACuGAuuGCCAuGGAuCCAu 23.51% 48.59%  5.07%  8.54% 9 8 6 7 Scn9a Sa Exon 12_T3 20 AGAAAACAAGGAGCCACGAAuG 20.49% 46.80%  3.51% 17.10% 10 9 10 2 SCN10A SaCas9 Scn10a Sa Exon 11_T1 31 CCCuGGAGCuGuCGAuGuCuCG 42.68% 0.00%  0.00%  0.39% 1 10 10 10 Scn10a Sa Exon 1_T7 32 uAGAuCCGuuCuACAGCACACA 40.36% 71.64%  6.10%  7.31% 2 2 3 5 Scn10a Sa Exon 8_T5 33 AGuGAGAGGAAAGCCCAAGCAA 35.32% 73.07%  7.37% 25.44% 3 1 2 2 Scn10a Sa Exon 12_T2 34 ACCuuuCCGGGCCCAAAGGGCA 32.36% 63.10% 11.88% 36.91% 4 3 1 1 Scn10a Sa Exon 14_T1 35 CuuuGACuGCAuCAuCGuCACu 31.27% 48.68%  3.67%  0.72% 5 8 5 9 Scn10a Sa Exon 14_T2 36 CACuuCuuCuGGAAAuAAuAGu 27.17% 39.07%  3.16%  3.03% 6 9 7 6 Scn10a Sa Exon 4_T5 37 AuuuuAGCGuCAuuACCCuGGC 25.46% 49.54%  1.86%  2.52% 7 7 9 8 Scn10a Sa Exon 1_T3 38 AACAACuuCCGuCGCuuuACuC 24.67% 57.17%  2.36%  2.87% 8 4 8 7 Scn10a Sa Exon 11_T2 39 GCCGAGAuAuCuCACuCCCuGA 23.87% 56.97%  4.61% 12.23% 9 5 4 4 Scn10a Sa Exon 11_T5 40 uGGuGuuCAuCuuCuCCAuGCC 23.03% 54.02%  3.32% 13.89% 10 6 6 3 Off-Target Evaluation of SpCas9 and SaCas9 gRNAs Targeted to SCN9A and SCN10A in iPSCs

Based on on-target efficacy in initial gRNA screens in iPSCs, 40 guides were also prioritized for an off-target evaluation. Specifically, ten guides from each of four categories were chosen: 1) ten gRNAs for SpCas9 targeting SCN9A, 2) ten gRNAs for SpCas9 targeting SCN10a, 3) ten gRNAs for SaCas9 targeting SCN9A, and 4) ten gRNAs for SaCas9 targeting SCN10a.

Of the 40 gRNAs included in the study, 29 gRNAs were categorized as “Tier 1” (Table 14), where no off-target sites included in the study entered statistical testing; these 29 gRNAs included 4 gRNAs where no off-target sites were predicted under the sequence similarity criteria. Based on this study, these 29 gRNAs are considered to have no evidence of off-target editing. In addition, seven gRNAs were categorized as “Tier 2” (Table 15), where at least one off-target site associated with that gRNAs may have entered statistical testing, but was not found to be statistically significant. These off-target profile of these gRNAs are considered to be inconclusive from this study. In addition, four gRNAs were categorized as “Tier 3” (Table 16), where at least one off-target site was found to have statistically significant off-target editing. These gRNAs were strongly deprioritized, based on these off-target editing results. All combinations of target genes (SCN9A or SCN10A) and enzymes (SpCas9 or SaCas9) were found to have at least 5 Tier 1 guides.

TABLE 14 29 gRNAs categorized as Tier 1 with no evidence of off-target editing # sites with any SEQ # tested evidence of gRNA Name ID NO: sites editing Scn9a Sp Exon 11_T9 1 44 0 Scn9a Sp Exon 11_T6 3 42 0 Scn9a Sp Exon 11_T14 4 83 0 Scn9a Sp Exon 11_T13 5 40 0 Scn9a Sp Exon 12_T11 9 42 0 Scn10a Sp Exon 10_T3 30 42 0 Scn10a Sp Exon 9_T2 22 54 0 Scn10a Sp Exon 14_T3 23 104 0 Scn10a Sp Exon 7_T6 25 117 0 Scn9a Sa Exon 12_T3 20 1 0 Scn9a Sa Exon 12_T1 16 1 0 Scn9a Sa Exon 12_T2 12 2 0 Scn9a Sa Exon 5_T6 13 1 0 Scn9a Sa Exon 9_T3 14 1 0 Scn9a Sa Exon 13_T3 15 2 0 Scn9a Sa Exon 11_T3 11 2 0 Scn9a Sa Exon 9_T2 18 1 0 Scn9a Sa Exon 15_T3 19 2 0 Scn10a Sa Exon 11_T5 40 2 0 Scn10a Sa Exon 8_T5 33 4 0 Scn10a Sa Exon 14_T1 35 2 0 Scn10a Sa Exon 14_T2 36 4 0 Scn10a Sa Exon 4_T5 37 1 0 Scn10a Sa Exon 11_T2 39 1 0 Scn10a Sp Exon 10_T7 24 68  1* Scn10a Sa Exon 11_T1 31 0 0 Scn10a Sa Exon 1_T7 32 0 0 Scn10a Sa Exon 12_T2 34 0 0 Scn10a Sa Exon 1_T3 38 0 0 *Scn10a Sp Exon 10_T7 had one site tested due to the 0.2% threshold requirement, but that site was ultimately excluded due to a germline mutation.

TABLE 15 Seven gRNAs categorized as Tier 2 with inconclusive off-target editing profiles SEQ T-test Chi-sq Treated Untreated # # gRNA name ID NO: Site ID pval pval (Indel %) (Indel %) mismatch gaps Scn10a Sp Exon 27 chr8: 3193432- 0.0981 0 0.89% 0.00% 2 1 11_T10 3193453 Scn9a Sa Exon 17 chr2: 165162747- 0.0715 0 0.67% 0.00% 1 1 7_T1 165162773 Scn9a Sp Exon 7 chr12: 51699567- 0.2293 0 0.62% 0.00% 0 1 7_T5 51699588 Scn9a Sp Exon 7 chr17: 49809267- 0.1707 0.0006 0.35% 0.00% 2 0 7_T5 49809289 Scn9a Sp Exon 2 chr5: 139982158- 0.0769 0 0.53% 0.00% 3 0 12_T25 139982180 Scn9a Sp Exon 6 chr18: 34382228- 0.0929 0.0140 0.17% 0.00% 3 0 14_T1 34382250 Scn9a Sp Exon 10 chr4: 41330183- 0.0841 0.0114 0.16% 0.00% 2 0 12_T17 41330205 Scn10a Sp Exon 29 chr1: 191298075- 0.1989 0.0107 0.15% 0.00% 3 0 4_T2 191298097

TABLE 16 Four gRNAs categorized as Tier 3 with off-target editing confirmed (p < .05) at one or more sites SEQ T-test Chi-sq Treated Untreated # # gRNA name ID NO: Site ID pval pval (Indel %) (Indel %) mismatch gaps Scn9a Sp Exon 8 chr12: 51663013- 0.020 3.52E−18 1.80% 0.00% 2 0 2_T10 51663035 Scn9a Sp Exon 8 chr4: 64749297- 0.030  6.26E−116 8.50% 0.00% 3 0 2_T10 64749319 Scn10a Sp Exon 21 chr15: 31179489- 0.050 1.74E−13 3.90% 0.00% 3 0 1_T15 31179511 Scn10a Sp Exon 21 chr16: 89566197- 0.080 3.48E−07 0.70% 0.00% 2 1 1_T15 89566218 Scn10a Sp Exon 26 chr2: 180785404- 0.010 9.20E−07 3.20% 0.00% 3 0 1_T30 180785426 Scn10a Sp Exon 26 chr5: 111925220- 0.090 7.59E−09 0.60% 0.00% 2 1 1_T30 111925241 Scn10a Sp Exon 28 chr1: 17434745- 0.000 7.48E−75 6.10% 0.00% 2 0 1_T17 17434767 Scn10a Sp Exon 28 chr9: 134428917- 0.030 0.000920453 0.40% 0.00% 3 0 1_T17 134428939

Other Embodiments

All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

From the above description, one of skill in the art can easily ascertain the essential characteristics of the present disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the disclosure to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

EQUIVALENTS

While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited. 

1. A gene editing system for modifying a sodium voltage-gated channel alpha subunit 9 (SCN9A) gene, the gene editing system comprising: (a) an RNA-guided DNA endonuclease or a first polynucleotide moiety, which comprises a first nucleotide sequence encoding the RNA-guided DNA endonuclease; and (b) a second polynucleotide moiety, which comprises a second nucleotide sequence encoding a guide RNA (gRNA), wherein the gRNA comprises a spacer sequence having the nucleotide sequence of any one of SEQ ID NOs: 1-20.
 2. The gene editing system of claim 1, wherein: (i) the RNA-guided DNA endonuclease of (a) is Staphylococcus pyogenes Cas9 (SpCas9); and (ii) the gRNA of (b) comprises a spacer sequence having the nucleotide sequence of any one of SEQ ID NOs: 1-10.
 3. (canceled)
 4. The gene editing system of claim 1, wherein: (i) the RNA-guided DNA endonuclease of (a) is Staphylococcus aureus Cas9 (SaCas9); and (ii) the gRNA of (b) comprises a spacer sequence having the nucleotide sequence of any one of SEQ ID NOs: 11-20.
 5. (canceled)
 6. The gene editing system of claim 1, wherein the gRNA of (b) further comprises a scaffold sequence, optionally wherein the scaffold sequence comprises the nucleotide sequence of SEQ ID NO:
 41. 7. (canceled)
 8. The gene editing system of claim 1, wherein the first nucleotide sequence encoding the RNA-guided endonuclease in (a) further comprises a nucleotide sequence encoding a nuclear localization signal (NLS), which is fused in-frame with the RNA-guided DNA endonuclease.
 9. (canceled)
 10. The gene editing system of claim 1, wherein the first polynucleotide moiety of (a) and the second polynucleotide moiety of (b) are of different polynucleotides, optionally wherein at least one of the different polynucleotides is a viral vector. 11.-12. (canceled)
 13. The gene editing system of claim 1, wherein a single polynucleotide comprises the first polynucleotide moiety of (a) and the second polynucleotide moiety of (b), optionally wherein the single polynucleotide is a viral vector. 14.-15. (canceled)
 16. A nucleic acid comprising the single polynucleotide of claim
 13. 17. A viral particle or a set of viral particles, which collectively comprises the gene editing system of claim
 1. 18. (canceled)
 19. A method of editing a sodium voltage-gated channel alpha subunit 9 (SCN9A) gene, the method comprising contacting a cell with: a gene editing system of claim
 1. 20. The method of claim 19, wherein the contacting step is performed by administering the gene editing system to a subject in need thereof. 21.-26. (canceled)
 27. The method of claim 19, further comprising administering the cell to a subject in need thereof, optionally wherein the cell is a stem cell.
 28. (canceled)
 29. A gene editing system for modifying a sodium voltage-gated channel alpha subunit 10 (SCNA10) gene, the gene editing system comprising: (a) an RNA-guided DNA endonuclease or a first polynucleotide moiety, which comprises a first nucleotide sequence encoding the RNA-guided DNA endonuclease; and (b) a second polynucleotide moiety, which comprises a second nucleotide sequence encoding a guide RNA (gRNA), wherein the gRNA comprises a spacer sequence having the nucleotide sequence of any one of SEQ ID NOs: 21-40.
 30. The gene editing system of claim 29, wherein: (i) the RNA-guided DNA endonuclease of (a) is Staphylococcus pyogenes Cas9 (SpCas9); and (ii) the gRNA of (b) comprises a spacer sequence having the nucleotide sequence of any one of SEQ ID NOs: 21-30.
 31. (canceled)
 32. The gene editing system of claim 29, wherein: (i) the RNA-guided DNA endonuclease of (a) is Staphylococcus aureus Cas9 (SaCas9); and (iii) the gRNA of (b) comprises a spacer sequence having the nucleotide sequence of any one of SEQ ID NOs: 31-40.
 33. The gene editing system of claim 29, wherein the gRNA of (b) further comprises a scaffold sequence, optionally wherein the scaffold sequence comprises the nucleotide sequence of SEQ ID NO:
 41. 34. (canceled)
 35. The gene editing system of claim 29, wherein the first nucleotide sequence encoding the RNA-guided endonuclease in (a) further comprises a nucleotide sequence encoding a nuclear localization signal (NLS), which is fused in-frame with the RNA-guided DNA endonuclease.
 36. (canceled)
 37. The gene editing system of claim 29, wherein the first polynucleotide moiety of (a) and the second polynucleotide moiety of (b) are of different polynucleotides, optionally wherein at least one of the different polynucleotides is a viral vector. 38.-39. (canceled)
 40. The gene editing system of claim 29, wherein a single polynucleotide comprises the first polynucleotide moiety of (a) and the second polynucleotide moiety of (b), optionally wherein the single polynucleotide is a viral vector. 41.-42. (canceled)
 43. A nucleic acid comprising the single polynucleotide of claim
 40. 44. A viral particle or a set of viral particles, which collectively comprises the gene editing system of claim
 29. 45. (canceled)
 46. A method of editing a sodium voltage-gated channel alpha subunit 10 (SCNA10) gene, the method comprising contacting a cell with: a gene editing system of claim
 29. 47. The method of claim 46, wherein the contacting step is performed by administering the gene editing system to a subject in need thereof. 48.-53. (canceled)
 54. The method of claim 46, further comprising administering the cell to a subject in need thereof, optionally wherein the cell is a stem cell.
 55. (canceled)
 56. A method of treating a subject having pain, the method comprising administering to the subject the gene-editing system of claim
 1. 57. (canceled)
 58. A method of treating a subject having pain, the method comprising administering to the subject the gene-editing system of claim
 29. 